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Imma Ferrer and Mike Thurman (1)Jerry A. Zweigenbaum, Melissa Churley and Phil Stremple (2)
(1) Center for Environmental Mass Spectrometry, University of Colorado, Boulder, CO, USA
(2) Agilent Technologies, Inc.
LC-MS and GC-MS Approaches for the Analysis of Emerging Contaminants in
Water Samples
OutlineOutline
1. LC-TOF-MS Approach for Analysis of EPA 1694 Pharmaceuticals in Water Samples.
2. LC-QqQ-MS Approach using Jet Stream as a highly sensitive methodology for the identification of Pharmaceuticals in the Environment.
3. Rapid Resolution chromatography: examples using UHPLC (1290 Infinity).
4. GC-MS-MS for the analysis of Phytoestrogens.
Instrumentation LC-TOF-MSAgilent Model 6220Specifications:4 GHz detector rate10,000 resolving power< 2 ppm accuracy
LC-QqQ-MSAgilent Model 64301290 UHPLC Infinity(Also Model 6460 with Jet Stream)
GC-MS-MS (QqQ)Agilent Model 7000
LC Conditions:• Columns:
Zorbax Eclipse Plus and SB:C18, 2.1 x 100 mm, 3.5 µm particles C18, 2.1 x 50 mm, 1.8 µm particlesC18 2.1 x 100 mm, 1.8 µm particles
• Mobile phaseAcetonitrile and water with 0.1 % formic acid.Gradient: 10 % ACN to 100 % in 1, 10 or 20 min.Flow = 0.6 mL/min and 1.2 mL/min.
Analytical ConditionsAnalytical ConditionsAnalytical ConditionsExtraction procedure (Water Samples):
• Gilson GX-271 ASPEC automated SPE system.• Solid-phase extraction with Oasis HLB cartridges.• 1 L sample to 1 mL extract or 100 mL sample to 500 µL extract.
Penicillin VDiltiazem
VirginiamycinPenicillin GDigoxin
TylosinOxolinic AcidDigoxigenin
TrimethoprimOxacillinDehydronifedipine
ThiabendazoleOfloxacinCotinine
SulfathiazoleNorfloxacinCodeine
SulfanilamideMiconazoleCloxacillin
SulfamethoxazoleLomefloxacinClarithromycin
SulfamethizoleLincomycinCiprofloxacin
SulfamethazineFluoxetineCefotaxime
SulfamerazineFlumequineCarbamazepine
SulfadimethoxineErythromycin AnhydrateCarbadox
SulfadiazineErythromycinCaffeine
SulfachloropyridazineEnrofloxacinAzithromycin
SarafloxacinDiphenhydramineAmpicillin
Roxithromycin1,7-dimethylxanthineAcetaminophen
Group 1
Ranitidine
Metformin
Cimetidine
Albuterol
Group 4
Warfarin
Triclocarban
Naproxen
Ibuprofen
Gemfibrozil
Group 3
EPA Method 1694
MacrolidesBenzomidazolesPenicillinesQuinolonesSulfonamidesPyrimidinesNSAIDs
Target Analytes
1. LC-TOF-MS for the Analysis of Pharmaceuticals
1. LC-TOF-MS for the Analysis of Pharmaceuticals
6x10
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
4.4
+ TIC Scan Mix_60_Oasis_Hot water_final volume 1mL_15uL.d
1 1
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Drinking Water Sample, spiked at 10 ug/L
6x10
0.2
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0.8
1
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1.6
1.8
2
2.2
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2.6
2.8
3
3.2
3.4
Cpd 1:+ ECC Scan Mix_60_Oasis_Hot water_final volume 1mL_15uL.d
.0
Counts vs. Acquisition Time (min)2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Ace
tam
inop
hen
Am
pici
llin
Sulfa
met
hoxa
zole
+ A
zith
rom
ycin
Caf
fein
e
Car
bam
azep
ine
Cef
otax
ime
Cip
roflo
xaci
n
Cla
rithr
omyc
in
Clo
xaci
llin
Cot
inin
e
Deh
ydro
nife
dipi
ne
Dig
oxig
enin
Dilt
iaze
m+
Dig
oxin
1,7-
dim
ethy
lxan
thin
e Dip
henh
ydra
min
e
Enro
floxa
cin
Eryt
hrom
ycin
Flum
equi
neFl
uoxe
tine
Linc
omyc
in+
Sulfa
diaz
ine
Lom
eflo
xaci
n
Mic
onaz
oleN
orflo
xaci
n+
Oflo
xaci
n
Oxa
cilli
n
Oxo
linic
Aci
d
Peni
cilli
n G
Rox
ithro
myc
in+
Peni
cilli
n V
Sara
floxa
cin
Sulfa
chlo
ropy
ridaz
ine
Sulfa
dim
etho
xine
Sulfa
mer
azin
e
Sulfa
met
hazi
ne
Sulfa
met
hizo
le
Sulfa
nila
mid
e
Thia
bend
azol
e
Trim
etho
prim
Tylo
sin
Virg
inia
myc
in
Wastewater SampleCotinineCaffeineCarbamazepineDiphenhydramineSulfamethoxazoleDEETThiabendazoleTrimethoprim
Screening and Use of DatabasesScreening and Use of DatabasesScreening and Use of Databases
5x10
0.5
1
1.5
2
2.5
3
+ EIC(194.0000) Scan Boulder WWO_19June08.d
1 1
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Non-target AnalysisNonNon--target Analysistarget AnalysisN
O NH2
C15H13N2O+
Exact Mass: 237.1022
Carbamazepine
NH2
C14H12N+
Exact Mass: 194.0964
Extracted Chromatogram m/z = 194
4x10
0
2
4
6
…237.1023
194.0963
274.2012255.1126
Counts vs. Mass-to-Charge (m/z)180 190 200 210 220 230 240 250 260 270 280 290 300
C15H15N2O20.83 ppm
N
O NH2
HO
C15H15N2O2+
Exact Mass: 255.1128
-18
2. LC-QqQ-MS for the Analysis of Pharmaceuticals
Jetstream Technology
Agilent Jet Stream gradient focusing technology delivers many more ions to the mass spectrometer while reducing the number of neutral solvent clusters. The result is stronger signals with lower RSD’s at the limit of detection.
4x10
0
0.2
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0.6
0.8
1
1.2
1.4
1.6
1.8
2
-ESI MRM Frag=75.0V [email protected] (205.0 -> 161.0) 1 ppb_All Groups+Extras.d
1 2 2 3
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Drinking water, spiked at 1 ppb concentrationC18 Eclipse Plus 2.1 x 100mm (3.5 μm) column
ComparisonQqQ 6460 Jet StreamQqQ 6410
50 times0.06 ppb3 ppbCarbamazepine
0.05 ppb
0.5 ppb
0.06 ppb
60 times3 ppbSulfadimethoxine
20 times10 ppbCaffeine
30 times2 ppbAcetaminophen
High sensitivity achieved with Jet Stream Technology
Comparison 6410 and 6460 with Jetstream
Instrumental LOD’s for Pharmaceuticals
Compound LOD Jetstream 6460 (µg/L)
1,7-Dimethylxanthine 0.6 Acetaminophen 0.06 Albuterol 0.02 Ampicillin 0.6 Azithromycin 6.0 Caffeine 0.5 Carbadox 0.3 Carbamazepine 0.06 Cefotaxime 2 Cimetidine 0.01 Ciprofloxacin 0.5 Clarithromycin 0.1 Cloxacillin 3.0 Codeine 0.3 Cotinine 0.05 Dehydronifedipine 0.03 Digoxigenin 0.4 Diltiazem 0.1 Diphenhydramine 0.05 Enrofloxacin 0.3 Erythromycin 0.3 Erythromycin Anhydrate
0.3
Flumequine 0.05 Fluoxetine 0.4 Gemfibrozil 0.1 Ibuprofen 7.0
After SPE concentration,more than 90% compounds
are detectable at< 1 ng/L in water
Compound LOD Jetstream 6460 (µg/L)
Lomefloxacin 0.4 Metformin 0.05 Miconazole 0.5 Naproxen 1.0 Norfloxacin 1.0 Ofloxacin 0.4 Oxolinic Acid 0.03 Penicillin G 1.0 Penicillin V 1.0 Ranitidine 0.05 Roxithromycin 0.5 Sarafloxacin 0.5 Sulfachloropyridazine 0.2 Sulfadiazine 0.5 Sulfadimethoxine 0.05 Sulfamerazine 0.1 Sulfamethazine 0.3 Sulfamethizole 0.3 Sulfamethoxazole 0.2 Sulfanilamide 4.0 Sulfathiazole 0.4 Thiabendazole 0.05 Triclocarban 0.1 Triclosan 1.0 Trimethoprim 0.5 Tylosin 6.0 Virginiamycin 0.4 Warfarin 0.1
Analysis of Wastewater Sample
Analysis of a Boulder Wastewater Outfall
for EPA 1694
7x10
0.5
1
1.5
2
2.5
3
3.5
+ TIC Scan Boulder WWO_19June08.d
1 1
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Carbamazepine
N
O NH2
m/z=194m/z=179
15.5 15.6 15.7 15.8 15.9 16 16.1 16.2 16
Cou
nts 5x10
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
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1.6
1.8
2
237.0 -> 194.0 , 237.0 -> 179.0
Ratio=12.5
+ MRM (15.596-16.139 min, 55 scans) (237.0 -> **) Boulder …
180 190 200 210 220 230 240
Cou
nts 6x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
194.0
179.0
237.0
+ MRM (14.321-14.770 min, 46 scans) (256.0 -> **) Boulder …
160 180 200 220 240 260
Cou
nts 5x10
0
0.10.2
0.30.4
0.5
0.60.7
0.8
0.91
1.11.2
1.3
1.41.5
167.0
152.0
256.0
14.1 14.2 14.3 14.4 14.514.6 14
Cou
nts 4x10
0
0.2
0.4
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0.8
1
1.2
1.4
1.6
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256.0 -> 167.0 , 256.0 -> 152.0
Ratio=30.0
14.1 14.2 14.3 14.4 14.514.6 14
Cou
nts 4x10
0
0.2
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0.8
1
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256.0 -> 167.0 , 256.0 -> 152.0
Ratio=30.0
Diphenhydramine
ON
CH3
CH3
m/z=167
m/z=152
3. Rapid Resolution Chromatography: UHPLC
Gradient: t0=10% ACNt3.4=10% ACNt20= 100% ACNt20.3=100% ACN
Flow-rate: 0.6 mL/minPressure: 750 Bar
C18 Eclipse Plus 2.1 x 100mm (1.8 μm) column
EPA Method 1694 Group 1 Analytes (46 pharmaceuticals) separationEPA Method 1694 Group 1 Analytes (46 pharmaceuticals) separation on on the the 1290 Infinity 1290 Infinity in half the time.in half the time.
Gradient: t0=10% ACNt1.7=10% ACNt10= 100% ACNt10.3=100% ACN
Flow-rate: 0.6 mL/minPressure: 375 Bar
C18 Eclipse Plus 2.1 x 50mm (1.8 μm) column
1 to 1.5 second peak widths for pharmaceuticals using LC1 to 1.5 second peak widths for pharmaceuticals using LC--MSMS--MS with a MS with a 55--ms dwell time and 20 points across the peak.ms dwell time and 20 points across the peak.
Faster and faster: “Gone in 60 seconds”
C18 Eclipse Plus 2.1 x 50mm (1.8 μm) column
Gradient: t0=10% ACNt1.5=100% ACN
Flow-rate: 1.2 mL/minPressure: 750 Bar
TIC in Black
0.9 sec
3x10
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3
+ESI MRM Frag=90.0V [email protected] (415.0000 -> 159.0000) Boulder effluent_C18_speed grad_15comp.d
1 1 2 2 3 3
Counts vs. Acquisition Time (min)0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45
Thia
bend
azol
e
Trim
etho
prim
Sulfa
met
hoxa
zole
Rox
yith
rom
ycin
Dilt
iaze
m
Cot
inin
e
Dip
henh
ydra
min
e
Fast analysis of pharmaceuticals in water following EPA Method 1694: Applications of UHPLC-MS/MSMichael Thurman
MS and Emerging Environmental Contaminants
3:30 pm
WOG pm
4. Analysis of Phytoestrogens by GC-MS-MS
Beneficial effects of flavonoids (water-soluble plant pigments derived from the 2-phenyl-1,4-benzopyrone structure) on humans have been reported.
Phytoestrogens are a group of nonsteroidalpolyphenolic compounds that occur naturally in plants (soy, alfalfa, clover…). They are used widely in several foods and beverages as food suplements.
Because of their structural similarity with estradiol(17-β-estradiol), have the ability to cause estrogenicor/and antiestrogenic effects.
Silk Heart Health
Analysis of Phytoestrogens in Soy Milk and Water
Extraction of Soy Milk (1mL) and Water Samples (100mL)With Ethyl Acetate (10mL)
Evaporation of EtAc to dryness
Derivatization with 200uL of 10% TMCS/BSTFA (1 hour at 60C)
Evaporation to dryness
Addition of 200uL ofBSTFA/Pyridine (5:1)
Injection of 1uL into GC-MS-MS
Derivatization of Phytoestrogens (USGS Method 64566-00015)Analytes studied:
• Biochanin A• Coumestrol• Daidzein• Equol• Formononetin• Genistein• Glycitein• Prunetin
O
OCH3
O
HO
OH
Biochanin A
Si(CH3)3
GC-MS-MS Parameters
Column: HP5 30m x 250 μm x 0.25 μm
15.5430010Ramp 2
5.5124040Ramp 1
11100Initial
Run Time (min)
Hold Time (min)
Value(ºC/min)
Rate(ºC/min)
2x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
+EI MRM CID@** (398.0000 -> 383.0000) Mix_Phytos_50ppb_MRM_gain30.D
1 1
Counts vs. Acquis ition Time (min)5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15
Dai
dzei
nG
enis
tein
Equo
l
Form
onon
etin
Bio
chan
in A
Prun
etin
Gly
cite
inC
oum
estr
ol
MRM chromatogram of the Phyto Mix
O
OSi(CH3)3O
(H3C)3SiO C21H26O4Si2•+
m/z 398
O
OSiOH
(H3C)3SiO
HO(H3C)3SiO C19H23O3Si2+
m/z 355
C20H23O4Si2+
m/z 383
-CH3
-C=O
OSi
Fragmentation pathway for Daidzein3x10
0
1
2
3
4
5
6
7
+EI Product Ion:1 (12.111-12.175 min, 4 scans) [email protected] (398.1 -> **) Daidzein_PI_39 …
383.2355.0
73.0 310.9
174.9
Counts vs. Mass-to-Charge (m/z)80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380
GC-MS-MS Fragmentation of Phytoestrogens
Analysis of Soy Milk
I.S.
Daidzein Genistein
Glycitein
Standard
Soy Milk Extract
471->399471->327
O
OSi(CH3)3
O
(H3C)3SiO
OSi(CH3)3
Genistein
m/z = 486
Backflush experiments
1
1 .5 1 2 1 2 .5 1 3 1 3 .5 1 4 1 4 .5 1 5
Without Backflush
With Backflush
ConclusionsConclusions
LC-TOF-MS useful for screening and non-target identification (e.g. degradation products).
LC-QqQ-MS offers high sensitivity for analysis of target pharmaceuticals (low ng/L detections).
Use of UHPLC to reduce total analysis time and enhances peak capacity on 1.8 μm columns.
GC-MS-MS is a robust and highly selective instrument for analysis of emerging contaminants in environmental samples.
AcknowledgmentsAcknowledgmentsAgilent Technologies for instrument support and providing standards.
Special Thanks to personnel in Little Falls (Wilmington, DE) and Santa Clara (CA) Agilent sites: Melissa Churley, Chin-Kai Meng, Harry Prest, Phil Stremple, Phil Wylie and Jerry Zweigenbaum.
Gilson Inc. for SPE support.
Larry Barber (from USGS) for sample collection in Boulder, CO.
University of Colorado for Lab space and support.
MRM transitions for Pharmaceuticals
I. Ferrer, E.M. Thurman and J.A. Zweigenbaum, App Note 5989-9665EN, 2008
Analysis of Phytoestrogens
LOD’s range from 1 ppb to 10 ppb
R2 > 0.999
