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copy2015 Waters Corporation 1
Analysis of Allergens in Perfumes Cosmetics and
Personal Care Products using UltraPerformance
Convergence Chromatographytrade (UPC2reg) with MS
Detection
Jane Cooper
Senior Applications Scientist
Chemical Materials Business Operations
Wilmslow UK
copy2015 Waters Corporation 2
Introduction
Background
ndashFragrances
ndashCosmetic Allergens
o Regulations
o Current methodologies
ndashUPC2
o Advantages over existing technologies
o Interfacing with MS detection
Summary of method development and results achieved using UPC2 with MS detection
Questions
copy2015 Waters Corporation 3
Fragrances
Fragrances are complex combinations of natural
andor man-made substances added to many
consumer products ndash to give them a distinctive smell
ndash impart a pleasant odour
ndash mask the inherent smell of some ingredients
ndash enhance the experience of the product user
ndash create important olfactory benefits that are
o ubiquitous tangible and valued
ndash used to communicate complex ideas such as
o creating mood
o signalling cleanliness
o freshness
o softness
o alleviating stress
o creating well-being
o trigger allure and attraction
copy2015 Waters Corporation 4
Fragrances
In most types of cosmetics and skin care products including
ndash Perfumes
ndash Shampoos
ndash Conditioners
ndash Moisturizers
ndash Facial cosmetics
ndash Deodorants
gt5000 different fragrances present
copy2015 Waters Corporation 5
Cosmetic Allergens
ndash An abnormal reaction of the body to a previously
encountered allergen introduced by
bull Inhalation
bull Ingestion
bull Skin contact
o Symptoms
bull Itchy eyes
bull runny nose
bull wheezing
bull skin rash
bull diarrhea
copy2015 Waters Corporation 6
Cosmetic Allergens
ndash Regulations
o EU Cosmetic Regulations (12232009)
bull Concentration exceeds 0001 in leave-on products
(10 mgkg) (eg a moisturiser) and 001 (100
mgkg) in rinse-off products (eg a shampoo)
copy2015 Waters Corporation 7
Current methodologies
GC-MS (~30-40 min)
HS-GC-MS (~30-40 min)
GC-GC-MS (~30-40 min)
LC-UV (~40 min)
Limitations Considerations of any method to be used for Allergen analysis include
ndash specificity
o allergens are small molecules (ions with low mz)
o many isobaric (same formula)
o with nonspecific fragment (close structure)
ndash resolution between analytes between isomers and with matrices components
ndash sensitivity at least 1 ppm (greater preferred)
copy2015 Waters Corporation 8
Allergens (structures)
Regulated Allergens2
1
Amyl Cinnamaldehyde
2
Benzyl alcohol
3
Cinnamyl alcohol
4
Citral CAS 122-40-7 (C14H18O) CAS 100-51-6 (C7H8O) CAS 104-54-1 (C9H10O) CAS 5392-40-5 (C10H16O)
5
Eugenol
6
Hydroxy-citronellal
7
Isoeugenol
8
Amyl cinnamyl alcohol
CAS 97-54-0 (C10H12O2) CAS 107-75-5 (C10H18O2) CAS97-54-1 (C10H12O2) CAS 101-85-9 (C14H20O)
9
Benzyl salycilate
10
Cinnamaldehyde
11
Coumarin
12
Geraniol
CAS 118-58-1 (C14H12O3) CAS 104-55-2 (C9H8O) CAS 94-64-5 (C9H6O2) CAS 106-24-1 (C10H18O)
13
Lyral
14
Anisyl alcohol
15
Benzyl cinnamate
16
Farnesol CAS 31906-04-4 (C13H22O2) CAS 105-13-5 (C7H8O2) CAS 103-41-3 (C16H14O2) CAS 4602-84-0 (C15H26O)
17
Lilial
18
Linalool
19
Benzyl benzoate
20
Citronellol
CAS 80-54-6 (C14H20O) CAS 78-70-6 (C10H18O) CAS 120-51-4 (C14H12O2) CAS 106-22-9 (C10H20O)
21
Hexyl cinnamaldehyde
22
Limonene
23
Methyl heptine carbonate
24
Alpha isomethyl ionone
CAS 101-86-0 (C15H20O) CAS 5989-27-5 (C10H16) CAS 111-12-6 (C9H14O2) CAS 127-51-5 (C14H22O)
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Regulated Cosmetic
allergens considered
as regulated under
current EU Cosmetic
Regulations (12232009)
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 2
Introduction
Background
ndashFragrances
ndashCosmetic Allergens
o Regulations
o Current methodologies
ndashUPC2
o Advantages over existing technologies
o Interfacing with MS detection
Summary of method development and results achieved using UPC2 with MS detection
Questions
copy2015 Waters Corporation 3
Fragrances
Fragrances are complex combinations of natural
andor man-made substances added to many
consumer products ndash to give them a distinctive smell
ndash impart a pleasant odour
ndash mask the inherent smell of some ingredients
ndash enhance the experience of the product user
ndash create important olfactory benefits that are
o ubiquitous tangible and valued
ndash used to communicate complex ideas such as
o creating mood
o signalling cleanliness
o freshness
o softness
o alleviating stress
o creating well-being
o trigger allure and attraction
copy2015 Waters Corporation 4
Fragrances
In most types of cosmetics and skin care products including
ndash Perfumes
ndash Shampoos
ndash Conditioners
ndash Moisturizers
ndash Facial cosmetics
ndash Deodorants
gt5000 different fragrances present
copy2015 Waters Corporation 5
Cosmetic Allergens
ndash An abnormal reaction of the body to a previously
encountered allergen introduced by
bull Inhalation
bull Ingestion
bull Skin contact
o Symptoms
bull Itchy eyes
bull runny nose
bull wheezing
bull skin rash
bull diarrhea
copy2015 Waters Corporation 6
Cosmetic Allergens
ndash Regulations
o EU Cosmetic Regulations (12232009)
bull Concentration exceeds 0001 in leave-on products
(10 mgkg) (eg a moisturiser) and 001 (100
mgkg) in rinse-off products (eg a shampoo)
copy2015 Waters Corporation 7
Current methodologies
GC-MS (~30-40 min)
HS-GC-MS (~30-40 min)
GC-GC-MS (~30-40 min)
LC-UV (~40 min)
Limitations Considerations of any method to be used for Allergen analysis include
ndash specificity
o allergens are small molecules (ions with low mz)
o many isobaric (same formula)
o with nonspecific fragment (close structure)
ndash resolution between analytes between isomers and with matrices components
ndash sensitivity at least 1 ppm (greater preferred)
copy2015 Waters Corporation 8
Allergens (structures)
Regulated Allergens2
1
Amyl Cinnamaldehyde
2
Benzyl alcohol
3
Cinnamyl alcohol
4
Citral CAS 122-40-7 (C14H18O) CAS 100-51-6 (C7H8O) CAS 104-54-1 (C9H10O) CAS 5392-40-5 (C10H16O)
5
Eugenol
6
Hydroxy-citronellal
7
Isoeugenol
8
Amyl cinnamyl alcohol
CAS 97-54-0 (C10H12O2) CAS 107-75-5 (C10H18O2) CAS97-54-1 (C10H12O2) CAS 101-85-9 (C14H20O)
9
Benzyl salycilate
10
Cinnamaldehyde
11
Coumarin
12
Geraniol
CAS 118-58-1 (C14H12O3) CAS 104-55-2 (C9H8O) CAS 94-64-5 (C9H6O2) CAS 106-24-1 (C10H18O)
13
Lyral
14
Anisyl alcohol
15
Benzyl cinnamate
16
Farnesol CAS 31906-04-4 (C13H22O2) CAS 105-13-5 (C7H8O2) CAS 103-41-3 (C16H14O2) CAS 4602-84-0 (C15H26O)
17
Lilial
18
Linalool
19
Benzyl benzoate
20
Citronellol
CAS 80-54-6 (C14H20O) CAS 78-70-6 (C10H18O) CAS 120-51-4 (C14H12O2) CAS 106-22-9 (C10H20O)
21
Hexyl cinnamaldehyde
22
Limonene
23
Methyl heptine carbonate
24
Alpha isomethyl ionone
CAS 101-86-0 (C15H20O) CAS 5989-27-5 (C10H16) CAS 111-12-6 (C9H14O2) CAS 127-51-5 (C14H22O)
[Type a quote from the document or
the summary of an interesting point
You can position the text box
anywhere in the document Use the
Text Box Tools tab to change the
formatting of the pull quote text box]
Regulated Cosmetic
allergens considered
as regulated under
current EU Cosmetic
Regulations (12232009)
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 3
Fragrances
Fragrances are complex combinations of natural
andor man-made substances added to many
consumer products ndash to give them a distinctive smell
ndash impart a pleasant odour
ndash mask the inherent smell of some ingredients
ndash enhance the experience of the product user
ndash create important olfactory benefits that are
o ubiquitous tangible and valued
ndash used to communicate complex ideas such as
o creating mood
o signalling cleanliness
o freshness
o softness
o alleviating stress
o creating well-being
o trigger allure and attraction
copy2015 Waters Corporation 4
Fragrances
In most types of cosmetics and skin care products including
ndash Perfumes
ndash Shampoos
ndash Conditioners
ndash Moisturizers
ndash Facial cosmetics
ndash Deodorants
gt5000 different fragrances present
copy2015 Waters Corporation 5
Cosmetic Allergens
ndash An abnormal reaction of the body to a previously
encountered allergen introduced by
bull Inhalation
bull Ingestion
bull Skin contact
o Symptoms
bull Itchy eyes
bull runny nose
bull wheezing
bull skin rash
bull diarrhea
copy2015 Waters Corporation 6
Cosmetic Allergens
ndash Regulations
o EU Cosmetic Regulations (12232009)
bull Concentration exceeds 0001 in leave-on products
(10 mgkg) (eg a moisturiser) and 001 (100
mgkg) in rinse-off products (eg a shampoo)
copy2015 Waters Corporation 7
Current methodologies
GC-MS (~30-40 min)
HS-GC-MS (~30-40 min)
GC-GC-MS (~30-40 min)
LC-UV (~40 min)
Limitations Considerations of any method to be used for Allergen analysis include
ndash specificity
o allergens are small molecules (ions with low mz)
o many isobaric (same formula)
o with nonspecific fragment (close structure)
ndash resolution between analytes between isomers and with matrices components
ndash sensitivity at least 1 ppm (greater preferred)
copy2015 Waters Corporation 8
Allergens (structures)
Regulated Allergens2
1
Amyl Cinnamaldehyde
2
Benzyl alcohol
3
Cinnamyl alcohol
4
Citral CAS 122-40-7 (C14H18O) CAS 100-51-6 (C7H8O) CAS 104-54-1 (C9H10O) CAS 5392-40-5 (C10H16O)
5
Eugenol
6
Hydroxy-citronellal
7
Isoeugenol
8
Amyl cinnamyl alcohol
CAS 97-54-0 (C10H12O2) CAS 107-75-5 (C10H18O2) CAS97-54-1 (C10H12O2) CAS 101-85-9 (C14H20O)
9
Benzyl salycilate
10
Cinnamaldehyde
11
Coumarin
12
Geraniol
CAS 118-58-1 (C14H12O3) CAS 104-55-2 (C9H8O) CAS 94-64-5 (C9H6O2) CAS 106-24-1 (C10H18O)
13
Lyral
14
Anisyl alcohol
15
Benzyl cinnamate
16
Farnesol CAS 31906-04-4 (C13H22O2) CAS 105-13-5 (C7H8O2) CAS 103-41-3 (C16H14O2) CAS 4602-84-0 (C15H26O)
17
Lilial
18
Linalool
19
Benzyl benzoate
20
Citronellol
CAS 80-54-6 (C14H20O) CAS 78-70-6 (C10H18O) CAS 120-51-4 (C14H12O2) CAS 106-22-9 (C10H20O)
21
Hexyl cinnamaldehyde
22
Limonene
23
Methyl heptine carbonate
24
Alpha isomethyl ionone
CAS 101-86-0 (C15H20O) CAS 5989-27-5 (C10H16) CAS 111-12-6 (C9H14O2) CAS 127-51-5 (C14H22O)
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Regulated Cosmetic
allergens considered
as regulated under
current EU Cosmetic
Regulations (12232009)
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 4
Fragrances
In most types of cosmetics and skin care products including
ndash Perfumes
ndash Shampoos
ndash Conditioners
ndash Moisturizers
ndash Facial cosmetics
ndash Deodorants
gt5000 different fragrances present
copy2015 Waters Corporation 5
Cosmetic Allergens
ndash An abnormal reaction of the body to a previously
encountered allergen introduced by
bull Inhalation
bull Ingestion
bull Skin contact
o Symptoms
bull Itchy eyes
bull runny nose
bull wheezing
bull skin rash
bull diarrhea
copy2015 Waters Corporation 6
Cosmetic Allergens
ndash Regulations
o EU Cosmetic Regulations (12232009)
bull Concentration exceeds 0001 in leave-on products
(10 mgkg) (eg a moisturiser) and 001 (100
mgkg) in rinse-off products (eg a shampoo)
copy2015 Waters Corporation 7
Current methodologies
GC-MS (~30-40 min)
HS-GC-MS (~30-40 min)
GC-GC-MS (~30-40 min)
LC-UV (~40 min)
Limitations Considerations of any method to be used for Allergen analysis include
ndash specificity
o allergens are small molecules (ions with low mz)
o many isobaric (same formula)
o with nonspecific fragment (close structure)
ndash resolution between analytes between isomers and with matrices components
ndash sensitivity at least 1 ppm (greater preferred)
copy2015 Waters Corporation 8
Allergens (structures)
Regulated Allergens2
1
Amyl Cinnamaldehyde
2
Benzyl alcohol
3
Cinnamyl alcohol
4
Citral CAS 122-40-7 (C14H18O) CAS 100-51-6 (C7H8O) CAS 104-54-1 (C9H10O) CAS 5392-40-5 (C10H16O)
5
Eugenol
6
Hydroxy-citronellal
7
Isoeugenol
8
Amyl cinnamyl alcohol
CAS 97-54-0 (C10H12O2) CAS 107-75-5 (C10H18O2) CAS97-54-1 (C10H12O2) CAS 101-85-9 (C14H20O)
9
Benzyl salycilate
10
Cinnamaldehyde
11
Coumarin
12
Geraniol
CAS 118-58-1 (C14H12O3) CAS 104-55-2 (C9H8O) CAS 94-64-5 (C9H6O2) CAS 106-24-1 (C10H18O)
13
Lyral
14
Anisyl alcohol
15
Benzyl cinnamate
16
Farnesol CAS 31906-04-4 (C13H22O2) CAS 105-13-5 (C7H8O2) CAS 103-41-3 (C16H14O2) CAS 4602-84-0 (C15H26O)
17
Lilial
18
Linalool
19
Benzyl benzoate
20
Citronellol
CAS 80-54-6 (C14H20O) CAS 78-70-6 (C10H18O) CAS 120-51-4 (C14H12O2) CAS 106-22-9 (C10H20O)
21
Hexyl cinnamaldehyde
22
Limonene
23
Methyl heptine carbonate
24
Alpha isomethyl ionone
CAS 101-86-0 (C15H20O) CAS 5989-27-5 (C10H16) CAS 111-12-6 (C9H14O2) CAS 127-51-5 (C14H22O)
[Type a quote from the document or
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Regulated Cosmetic
allergens considered
as regulated under
current EU Cosmetic
Regulations (12232009)
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 5
Cosmetic Allergens
ndash An abnormal reaction of the body to a previously
encountered allergen introduced by
bull Inhalation
bull Ingestion
bull Skin contact
o Symptoms
bull Itchy eyes
bull runny nose
bull wheezing
bull skin rash
bull diarrhea
copy2015 Waters Corporation 6
Cosmetic Allergens
ndash Regulations
o EU Cosmetic Regulations (12232009)
bull Concentration exceeds 0001 in leave-on products
(10 mgkg) (eg a moisturiser) and 001 (100
mgkg) in rinse-off products (eg a shampoo)
copy2015 Waters Corporation 7
Current methodologies
GC-MS (~30-40 min)
HS-GC-MS (~30-40 min)
GC-GC-MS (~30-40 min)
LC-UV (~40 min)
Limitations Considerations of any method to be used for Allergen analysis include
ndash specificity
o allergens are small molecules (ions with low mz)
o many isobaric (same formula)
o with nonspecific fragment (close structure)
ndash resolution between analytes between isomers and with matrices components
ndash sensitivity at least 1 ppm (greater preferred)
copy2015 Waters Corporation 8
Allergens (structures)
Regulated Allergens2
1
Amyl Cinnamaldehyde
2
Benzyl alcohol
3
Cinnamyl alcohol
4
Citral CAS 122-40-7 (C14H18O) CAS 100-51-6 (C7H8O) CAS 104-54-1 (C9H10O) CAS 5392-40-5 (C10H16O)
5
Eugenol
6
Hydroxy-citronellal
7
Isoeugenol
8
Amyl cinnamyl alcohol
CAS 97-54-0 (C10H12O2) CAS 107-75-5 (C10H18O2) CAS97-54-1 (C10H12O2) CAS 101-85-9 (C14H20O)
9
Benzyl salycilate
10
Cinnamaldehyde
11
Coumarin
12
Geraniol
CAS 118-58-1 (C14H12O3) CAS 104-55-2 (C9H8O) CAS 94-64-5 (C9H6O2) CAS 106-24-1 (C10H18O)
13
Lyral
14
Anisyl alcohol
15
Benzyl cinnamate
16
Farnesol CAS 31906-04-4 (C13H22O2) CAS 105-13-5 (C7H8O2) CAS 103-41-3 (C16H14O2) CAS 4602-84-0 (C15H26O)
17
Lilial
18
Linalool
19
Benzyl benzoate
20
Citronellol
CAS 80-54-6 (C14H20O) CAS 78-70-6 (C10H18O) CAS 120-51-4 (C14H12O2) CAS 106-22-9 (C10H20O)
21
Hexyl cinnamaldehyde
22
Limonene
23
Methyl heptine carbonate
24
Alpha isomethyl ionone
CAS 101-86-0 (C15H20O) CAS 5989-27-5 (C10H16) CAS 111-12-6 (C9H14O2) CAS 127-51-5 (C14H22O)
[Type a quote from the document or
the summary of an interesting point
You can position the text box
anywhere in the document Use the
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Regulated Cosmetic
allergens considered
as regulated under
current EU Cosmetic
Regulations (12232009)
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 6
Cosmetic Allergens
ndash Regulations
o EU Cosmetic Regulations (12232009)
bull Concentration exceeds 0001 in leave-on products
(10 mgkg) (eg a moisturiser) and 001 (100
mgkg) in rinse-off products (eg a shampoo)
copy2015 Waters Corporation 7
Current methodologies
GC-MS (~30-40 min)
HS-GC-MS (~30-40 min)
GC-GC-MS (~30-40 min)
LC-UV (~40 min)
Limitations Considerations of any method to be used for Allergen analysis include
ndash specificity
o allergens are small molecules (ions with low mz)
o many isobaric (same formula)
o with nonspecific fragment (close structure)
ndash resolution between analytes between isomers and with matrices components
ndash sensitivity at least 1 ppm (greater preferred)
copy2015 Waters Corporation 8
Allergens (structures)
Regulated Allergens2
1
Amyl Cinnamaldehyde
2
Benzyl alcohol
3
Cinnamyl alcohol
4
Citral CAS 122-40-7 (C14H18O) CAS 100-51-6 (C7H8O) CAS 104-54-1 (C9H10O) CAS 5392-40-5 (C10H16O)
5
Eugenol
6
Hydroxy-citronellal
7
Isoeugenol
8
Amyl cinnamyl alcohol
CAS 97-54-0 (C10H12O2) CAS 107-75-5 (C10H18O2) CAS97-54-1 (C10H12O2) CAS 101-85-9 (C14H20O)
9
Benzyl salycilate
10
Cinnamaldehyde
11
Coumarin
12
Geraniol
CAS 118-58-1 (C14H12O3) CAS 104-55-2 (C9H8O) CAS 94-64-5 (C9H6O2) CAS 106-24-1 (C10H18O)
13
Lyral
14
Anisyl alcohol
15
Benzyl cinnamate
16
Farnesol CAS 31906-04-4 (C13H22O2) CAS 105-13-5 (C7H8O2) CAS 103-41-3 (C16H14O2) CAS 4602-84-0 (C15H26O)
17
Lilial
18
Linalool
19
Benzyl benzoate
20
Citronellol
CAS 80-54-6 (C14H20O) CAS 78-70-6 (C10H18O) CAS 120-51-4 (C14H12O2) CAS 106-22-9 (C10H20O)
21
Hexyl cinnamaldehyde
22
Limonene
23
Methyl heptine carbonate
24
Alpha isomethyl ionone
CAS 101-86-0 (C15H20O) CAS 5989-27-5 (C10H16) CAS 111-12-6 (C9H14O2) CAS 127-51-5 (C14H22O)
[Type a quote from the document or
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Regulated Cosmetic
allergens considered
as regulated under
current EU Cosmetic
Regulations (12232009)
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 7
Current methodologies
GC-MS (~30-40 min)
HS-GC-MS (~30-40 min)
GC-GC-MS (~30-40 min)
LC-UV (~40 min)
Limitations Considerations of any method to be used for Allergen analysis include
ndash specificity
o allergens are small molecules (ions with low mz)
o many isobaric (same formula)
o with nonspecific fragment (close structure)
ndash resolution between analytes between isomers and with matrices components
ndash sensitivity at least 1 ppm (greater preferred)
copy2015 Waters Corporation 8
Allergens (structures)
Regulated Allergens2
1
Amyl Cinnamaldehyde
2
Benzyl alcohol
3
Cinnamyl alcohol
4
Citral CAS 122-40-7 (C14H18O) CAS 100-51-6 (C7H8O) CAS 104-54-1 (C9H10O) CAS 5392-40-5 (C10H16O)
5
Eugenol
6
Hydroxy-citronellal
7
Isoeugenol
8
Amyl cinnamyl alcohol
CAS 97-54-0 (C10H12O2) CAS 107-75-5 (C10H18O2) CAS97-54-1 (C10H12O2) CAS 101-85-9 (C14H20O)
9
Benzyl salycilate
10
Cinnamaldehyde
11
Coumarin
12
Geraniol
CAS 118-58-1 (C14H12O3) CAS 104-55-2 (C9H8O) CAS 94-64-5 (C9H6O2) CAS 106-24-1 (C10H18O)
13
Lyral
14
Anisyl alcohol
15
Benzyl cinnamate
16
Farnesol CAS 31906-04-4 (C13H22O2) CAS 105-13-5 (C7H8O2) CAS 103-41-3 (C16H14O2) CAS 4602-84-0 (C15H26O)
17
Lilial
18
Linalool
19
Benzyl benzoate
20
Citronellol
CAS 80-54-6 (C14H20O) CAS 78-70-6 (C10H18O) CAS 120-51-4 (C14H12O2) CAS 106-22-9 (C10H20O)
21
Hexyl cinnamaldehyde
22
Limonene
23
Methyl heptine carbonate
24
Alpha isomethyl ionone
CAS 101-86-0 (C15H20O) CAS 5989-27-5 (C10H16) CAS 111-12-6 (C9H14O2) CAS 127-51-5 (C14H22O)
[Type a quote from the document or
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Regulated Cosmetic
allergens considered
as regulated under
current EU Cosmetic
Regulations (12232009)
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 8
Allergens (structures)
Regulated Allergens2
1
Amyl Cinnamaldehyde
2
Benzyl alcohol
3
Cinnamyl alcohol
4
Citral CAS 122-40-7 (C14H18O) CAS 100-51-6 (C7H8O) CAS 104-54-1 (C9H10O) CAS 5392-40-5 (C10H16O)
5
Eugenol
6
Hydroxy-citronellal
7
Isoeugenol
8
Amyl cinnamyl alcohol
CAS 97-54-0 (C10H12O2) CAS 107-75-5 (C10H18O2) CAS97-54-1 (C10H12O2) CAS 101-85-9 (C14H20O)
9
Benzyl salycilate
10
Cinnamaldehyde
11
Coumarin
12
Geraniol
CAS 118-58-1 (C14H12O3) CAS 104-55-2 (C9H8O) CAS 94-64-5 (C9H6O2) CAS 106-24-1 (C10H18O)
13
Lyral
14
Anisyl alcohol
15
Benzyl cinnamate
16
Farnesol CAS 31906-04-4 (C13H22O2) CAS 105-13-5 (C7H8O2) CAS 103-41-3 (C16H14O2) CAS 4602-84-0 (C15H26O)
17
Lilial
18
Linalool
19
Benzyl benzoate
20
Citronellol
CAS 80-54-6 (C14H20O) CAS 78-70-6 (C10H18O) CAS 120-51-4 (C14H12O2) CAS 106-22-9 (C10H20O)
21
Hexyl cinnamaldehyde
22
Limonene
23
Methyl heptine carbonate
24
Alpha isomethyl ionone
CAS 101-86-0 (C15H20O) CAS 5989-27-5 (C10H16) CAS 111-12-6 (C9H14O2) CAS 127-51-5 (C14H22O)
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Regulated Cosmetic
allergens considered
as regulated under
current EU Cosmetic
Regulations (12232009)
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 9
Additional compounds considered (structures)
Six additional compounds were
also analyzed
Four cosmetic allergens not
yet regulated under current
EU Cosmetic Regulations
(12232009)
Two compounds that are
potential carcinogens
(methyl eugenol and 4-
allyl anisole)
Additional compounds considered
25
Atranol
26
Chloratranol
27
Methyl-2-nonynoate CAS 526-37-4 (C8H8O3) CAS 57074-21-2 (C8H7ClO3) CAS 111-80-8 (C10H16O2)
28
Methyl eugenol
29
Phenylacetaldehyde
30
4-Allyl anisole
CAS 93-15-2 (C11H14O2)
CAS 122-78-1 (C8H8O)
CAS 140-67-0 (C10H12O)
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 10
Allergens
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 11
Convergence Chromatography (UPC2)
Based on the principles of normal-phase LC
Uses CO2 as the primary mobile phase
Choice to add a co-solvent
With the ease-of-use of reversed-phase LC
With the separation power of Normal Phase
chromatography
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 12
Convergence Chromatography (UPC2) Analyses a Diverse range of compounds
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 13
The UPC2 Advantage
ndash Breakthrough technique
o Expands the boundaries of reversed phase LC and GC
o Replacement for normal phase LC
ndash Facilitates analysis of mixtures with components of wide polarity
range
ndash Separate and characterize compounds that challenge GC and LC
ndash Eliminate need to work with toxic organic solvents (greener
technology)
Why UPC2 over existing technology
radic
radic
radic
radic
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 14
Sample manager Isocratic
Solvent Manager
QDa or MS
Binary Solvent manager
Convergence Manager
PDA Detector
ELSD
Column Manager
(30S)
Convergence Chromatography (UPC2)
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 15
Convergence Chromatography (UPC2)
Xevo TQD UPC2
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 16
Interfacing UPC2 with MS detection
MS splitter
From the Column manager or PDA
Isocratic Solvent Manager
To the convergence Manager
To the MS
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 17
Universal Source Platform
Electrospray
Ionization (ESI)
Atmospheric
Pressure
Chemical
Ionization (APCI)
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 18
Interfacing UPC2 with MS detection
Electrospray Ionization (ESI)
ndash An electrically charged field is used to generate charged droplets
then analyte ions are formed by evaporation prior to MS analysis
ndash The addition of a protonation source such as formic acid to the
mobile phases to enhance ionization and increase sensitivity
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 19
Interfacing UPC2 with MS detection
Atmospheric Pressure Chemical Ionization (APCI)
ndash The solvent present acts as chemical ionization reagent gas in order
to ionize the sample
ndash The sample in the gas phases is then ionized by corona discharge
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 20
Parameters considered
UPC2 column
Modifier
Pressure
Gradient
Flow rate
Sample diluent
Temperature
Make-up solvent flow
Ionization mode
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 21
Preliminary Results
Initial screen of 8 UPC2 columns
o ACQUITY UPC2 BEH
o ACQUITY UPC2 CSH Flourophenyl
o ACQUITY UPC2 2-EP
o ACQUITY UPC2 HSS C18 SB
o ACQUITY UPC2 Torus 2-PIC (2-picolylamine)
o ACQUITY UPC2 Torus 1-AA (1-aminoanthracene)
o ACQUITY UPC2 Torus Diol (High Density Diol)
o ACQUITY UPC2 Torus DEA (Diethylamine)
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 22
BEH 2-Ethylpyridine [2-EP]
ndash Exhibits good retention peak shape and selectivity properties without the use of additives
BEH
ndash Heightens interaction with polar analyte functional groups when compared to the less polar surface of BEH 2-EP
CSH Fluoro-Phenyl
ndash Provides good retention for weak bases and alternate elution orders for acidic and neutral compounds via a significantly different selectivity
Viridisreg Hybrid SFC Chemistries
Two particles Ethylene Bridged Hybrid (BEH) in 17 amp 5 microm Charged Surface Hybrid (CSH) in 17 amp 5 microm
Multiple dimensions (5 microm only)
50 75 100 150 250 mm length 21 30 46 mm ID
VanGuardtrade pre-columns
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 23
Four Unique Column Chemistries
ACQUITY UPC2 Torus 2-PIC
ndash 2-picolylamine
ACQUITY UPC2 Torus DEA
ndash Diethylamine
ACQUITY UPC2 Torus DIOL
ndash High density diol
ACQUITY UPC2 Torus 1-AA
ndash 1-aminoanthracene
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 24
Preliminary Results
UPC2 conditions (column screening conditions)
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Columns BEH CSH Flourophenyl 2-EP 2-PIC 1-AA
Diol DEA HSS C18 SB
Mobile phase A CO2
Mobile phase B Ethanol
Isocratic Solvent Manager Methanol (05 mLmin)
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 25
Column screening
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 26
Modifier solvents screened
o Ethanol
o Methanol
o Isopropyl alcohol (IPA)
o Acetonitrile
Preliminary Results
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 27
Modifier screening
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 28
Time100 200 300 400 500 600 700
5000
10000
15000
20000462
Methods
UPC2 conditions
CCM back pressure 1500 psi
Sample temp 15 oC
Column temp 60 oC
Injection volume 3 microL
Column ACQUITY UPC2 C18 HSS
30 mm x 150 mm 18 microm
Mobile phase A CO2
Mobile phase B Methanol (01 Formic Acid)
Isocratic Solvent Manager Methanol (04 mLmin)
Time
(min)
Flow
(mLmin)
A
(CO2) B Curve
1 Initial 15 995 05 Initial
2 45 15 854 146 6
3 46 15 80 20 6
4 5 15 80 20 6
5 505 15 995 05 6
6 7 15 995 05 6
20 CO2 B
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 29
Method Information Detectors
MS system Xevo TQD
Ionization mode APCI (positive and negative)
Corona voltage 10 microA
Source temp 150 oC
APCI Probe temp 600 oC
Desolvation gas 1000 Lhr
Cone gas 15 Lhr
Acquisition Multiple Reaction Monitoring
(MRM)
Method Information MS
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 30
MRM chromatograms
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 31
Method Information Detectors
Method Information MS
No Chemical Substance Retention time
(min) isomers
APCI (+-)
Cone Voltage
(V) Transition
Collision energy
1 Amyl Cinnamaldehyde 184 + 30 2030 gt 1290 18
2030 gt 1470 16
2 Benzyl alcohol 186 + 8 1550 gt 910 8
1550 gt 1230 4
3 Cinnamyl alcohol 278 + 25 1330 gt 1850 18
4 Citral 158 + 15 1530 gt 690 6
1530 gt 950 15
5 Eugenol 168 + 20 1651 gt 1240 20
1651 gt 1371 12
6 Hydroxy-citronellal 337 + 18 1710 gt 1110 15
1710 gt 1530 10
7 Isoeugenol 190 + 25 1651 gt 1050 20
1651 gt 1330 20
8 Amyl cinnamyl alcohol 284 + 25 1870 gt 1170
20
1870 gt 1310 16
9 Benzyl salycilate 186 + 15 2290 gt 910 12
2290 gt 1510 12
10 Cinnamaldehyde 175 + 25 1330 gt 550 18
1330 gt 1150 14
11 Coumarine 252 + 40 1470 gt 910 28
1470 gt 1030 23
12 Geraniol 159 + 20 1370 gt 810 14
1370 gt 950 16
13 Lyral 324 + 20 1930 gt 1110 18
1930 gt 1750 12
14 Anisyl alcohol 279 + 40 1210 gt 770 25
1210 gt 780 25
15 Benzyl cinnamate 231 + 25 2210 gt 1050 6
2210 gt 1930 8
16 Farnesol 261276283 + 25 2051 gt 1090 20
2051 gt 1210 20
17 Lilial 231 + 10 2212 gt 909 30
18 Linalool 223 + 20 1370 gt 810 20
1370 gt 950 20
19 Benzyl benzoate 187 + 8 2130 gt 910 8
20 Citronellol 219 + 18 1571 gt 570 10
1571 gt 830 10
21 Hexyl cinnamaldehyde 194 + 30 2174 gt 129 20
2174 gt 147 14
22 Limonene 067 + 20 1370 gt 810 14
1370 gt 950 16
23 Methyl heptine carbonate
072 + 30 1550 gt 670 24
1550 gt 1230 15
24 Alpha isomethyl ionone 165 + 20 2072 gt 1111 20
2072 gt 1231 20
25 Atranol 457 - 18 1510 gt 7894 20
1510 gt 12309 20
26 Chloratranol 290 - 18 1850 gt 12117 20
1850 gt 15699 20
27 Methyl-2-nonynoate 153 + 34 1530 gt 429 22
1530 gt 970 16
28 Methyl eugenol 178 + 25 1790 gt 138 16
1790 gt 164 14
29 Phenylacetaldehyde 070 + 2 1210 gt 569 4
1210 gt 890 10
30 4-Allyl anisole 252 + 30 1469 gt 769 28
1469 gt 909 32
Cosmetic allergens
expected retention times
ionization mode cone
voltages MRM transitions
and associated collision
energy values refer to the quantitation transition
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 32
Method Information Detectors
Method Information MS
Benzyl salycilate
CAS 118-58-1 (C14H12O3)
Mass = 2282433
GCMS ndash typically 91 mz (EI)
APCI - softer ionization technique
MRM transitions (Precursor gt product ion)
2290 gt 910 mz
2290 gt 1510 mz
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 33
Calibration Curve
Compound name Cinnamyl Alcohol
Correlation coefficient r = 0999967 r^2 = 0999935
Calibration curve 931955 x + -86289
Response type External Std Area
Curve type Linear Origin Include Weighting Null Axis trans None
Conc-00 20 40 60 80 100 120 140 160 180 200 220 240
Re
sp
on
se
-0
5000
10000
15000
20000
025 to 25ppm
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 34
Preliminary Results
Sample analysis
ndash Perfume
(10 mgKg)
100 microL perfume + 900 microl
(methanol + 20 mM
Ammonium hydrogen
carbonate)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 35
Preliminary Results
Sample analysis
ndash Shampoo
(100 mgKg)
ndash 02 g sample add 25 mL
water and 25 mL
(methanol + 20 mM
Ammonium hydrogen
carbonate)
ndash Mixture vortexed for 2
minutes (1600 rpm)
ndash Mixture further mixed in an
ultrasonic bath for 30
minutes
ndash Approximately 1 mL of
extract centrifuged for 5
min (10000 rpm)
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
-000 100 200 300 400
0
Time100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
100 200 300 400
2
100 200 300 400
0
100 200 300 400
0
100 200 300 400
0
Time200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
0
200 300 400 500
3
Methyl eugenol
Cinnamaldehyde
Eugenol
Alpha isomethyl
ionone
Geraniol
Methyl-2-nonynoate
Citral
Limonene
Phenylacetaldehyde
Methyl heptinecarbonate
Hydroxy-citronellal
Lyral
Amyl cinnamylalcohol
Cinnamylalcohol
Anisylalcohol
Coumarine
4-Allyl anisole
Farnesol
Lilial
Benzyl cinnamate
Linalool
Citronellol
Hexylcinnamaldehyde
Isoeugenol
Amyl Cinnamaldehyde
Benzyl alcohol
Benzyl salycilate
Benzyl benzoate
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 36
Farnesol
2-trans6-cis-Farnesol Synonym 2E6Z-Farnesol
2-cis6-trans-Farnesol Synonym 2Z6E-Farnesol
transtrans-Farnesol Synonym
2E6E-Farnesol
ciscis-Farnesol Synonym 2Z6Z-Farnesol
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 37
Time230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
230 240 250 260 270 280 290 300 310 320
-0
100
Farnesol in shampoo
Farnesol (shampoo)
05 ppm Farnasol standard (equivalent to 125 mgKg)
Farnesol (shampoo fortified at 10 mgKg)
Major = transtrans Minor = cistrans and transcis Often not seen = ciscis
Transitions 2051 gt 1090 mz 2051 gt 1210 mz
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 38
Chloratranol and Atranol
Time100 200 300 400 500 600 700 800 900
0
100 200 300 400 500 600 700 800 900
0
457
421
Atranol
Chloratranol
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 39
Conclusion
Separation by UPC2 is an ideal alternative to both HPLC and GC analysis
Ability to run LC and GC amenable compounds in single analysis
Fast 7 minute analysis of the 24 regulated allergens and 6
additional compounds containing ndash different classes of compounds
ndash different polarities
UPC2 with MS detection offers an orthogonal technique which
enables greater selectivity and specificity compared to either HPLC or GC analysis alone
The developed 7 minute UPC2 method is greater than 6 times
faster than existing HPLC and GC methods with 95 less solvent usage than existing HPLC method
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 40
Acknowledgments
Waters (France)
ndash Steacutephane Dubant
ndash Freddy Delsenne
Waters (Wilmsow)
ndash Peter Hancock
ndash Michael Jones
ERINI
ndash Ceacuteline Roy
Yves Rocher
ndash Beatrice Grimaud
ndash Isabelle Dubrulle
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 41
Application Note
720005553en (available on wwwwaterscom)
copy2015 Waters Corporation 42
Any Questions
copy2015 Waters Corporation 42
Any Questions
