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©2015 Waters Corporation 1
Transforming Sample Preparation
Doug McCabe, Waters Corporation
HPLC 2015, Geneva
©2015 Waters Corporation 2
Transforming Sample Preparation
When you think about doing sample preparation, would
you rather do this?
Start
End
©2015 Waters Corporation 3
Transforming Sample Preparation
Or this?
Sta
rt
En
d
©2015 Waters Corporation 4
Agenda
The importance of sample preparation and industry trends
Simplifying Sample Preparation
Introducing Oasis PRiME HLB
Applications and comparisons
Conclusions
©2015 Waters Corporation 5
Why Do Sample Preparation?
Need to remove matrix interferences – Increase signal to noise by simplifying the chromatographic separation
o Enables better, more consistent quantitation
– Reduce variability in analytical results due to matrix inconsistencies
o Higher, more consistent recovery
o Minimize matrix effects
o Less rework
– Increase column lifetime
o Fewer columns need to be replaced
– Reduces system downtime
o Less time spent with wrenches or waiting for service
Need to concentrate analyte of interest – Present in low levels = difficult to quantitate
Need to transfer (extract) analytes of interest into a solution that can be tested – Meat, fish and milk
But How?
©2015 Waters Corporation 6
How can I turn this?
Into this?
Sample Preparation
©2015 Waters Corporation 7
Sample Preparation Techniques: Which One?
How do you choose a technique to clean up complex sample
matrices?
– Filtration / Dilution
– Protein precipitation (PPT)
– Liquid-liquid extraction (LLE)
– Solid-supported liquid-liquid extraction (SSLE)
– Solid-phase extraction (SPE)
Objectives: Simplest technique
Fastest preparation procedure Cleanest extracts
“Good Enough” Sample Preparation
©2015 Waters Corporation 8
How Good is Good Enough?
Fast gradients are common and help increase throughput, but…
It is important to be able to accurately quantify drugs and metabolites during the discovery stage and preclinical assessments of candidate drugs (eg. for MIST assessment). The method needs to be fast, robust and applicable to diverse drug candidates and metabolites.
10 parent drugs
10 metabolites
©2015 Waters Corporation 9
Phospholipid Build Up Can Lead to Matrix Effects and Unpredictable Results
Using protein precipitation, residual phospholipids build up on the column during the processing of one analytical batch (50 samples total) The bottom chromatogram shows the earliest and latest eluting parent compounds superimposed over the phospholipid trace, illustrating co-elution. This may cause matrix effects. Also note the elution of a significant phospholipid peak at the end of the gradient re-equilibration. This may cause matrix effects and a decrease in robustness.
Application Note: Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies, 720004453en
Application Note: Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies, 720004453en Danaceau et al. 2014 Bioanalysis 6(6) 761-771
©2015 Waters Corporation 10
Phospholipid Build Up Can Lead to Matrix Effects and Unpredictable Results
Using protein precipitation, remaining phospholipids build up on the column during the processing of one analytical batch. The bottom chromatogram shows the earliest and latest eluting parent compounds superimposed over the phospholipid trace, illustrating co-elution. This may cause matrix effects. Also note the elution of a significant phospholipid peak at the end of the gradient re-equilibration. This may cause matrix effects and a decrease in robustness.
Application Note: Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies, 720004453en
Is it possible to solve this problem,
without a lot of extra work and
effort?
©2015 Waters Corporation 11
Agenda
The importance of sample preparation and industry trends
Simplifying Sample Preparation
©2015 Waters Corporation 12
History – Making SPE Simple
Since 1996, Waters goal was to simplify complex sample
preparation methods with generic sorbent & protocols
– Oasis HLB introduced in 1996 as the first water-wettable polymeric
SPE into the market
– Gold standard SPE sorbent in sample prep market for the last 18
years
– Sample preparation technique of choice for LC-MS due to cleanliness
of extracts and universal applicability.
– Well understood SPE technology
– 5 step generic protocol
Condition
Equilibrate
Wash
Elute
Load
• 200 µL Methanol (MeOH)
• 200 µL H2O
• 200 µL 5% MeOH in H2O
• 25 µL*2 MeOH
• 200 µL spiked dilute plasma (100 µL plasma + 100µL 4% H3PO4)
©2015 Waters Corporation 13
Oasis HLB: First Water-Wettable Sorbent
Hydrophilic
monomer
Lipophilic
monomer
N O
Reversed-phase retention
Hydrophilic-Lipophilic Balanced Copolymer
Retention of polar compounds
C18 silica-based sorbents and non water-wettable polymeric sorbents dewet if allowed to dry
Oasis sorbents do not exhibit this undesirable behavior
©2015 Waters Corporation 14
Oasis uElution Protocol Comparison: Standard vs. Simplified
Condition
Equilibrate
Wash
• 200 µL 5% MeOH in H2O
Elute
• 25 µL*2 MeOH
Load
• 200 µL spiked dilute plasma (100 µL plasma + 100µL 4% H3PO4)
• 200 µL Methanol (MeOH)
• 200 µL H2O
• 200 µL 5% MeOH in H2O
• 25 µL*2 MeOH
• 200 µL spiked dilute plasma (100 µL plasma + 100µL 4% H3PO4)
Standard Simplified
©2015 Waters Corporation 15
Oasis HLB vs. Competitor Recovery: Simplified Protocol
Oasis HLB works well with simplified 3-step (Load-Wash-Elute) protocol Average 3 Step Recoveries = 83±1.7% (Average 5 Step Recoveries = 90±4.5%)
Competitor Sµ plate does not work with simplified 3-step (Load-Wash-Elute)
protocol Comparative separations may not be representative of all applications.
0102030405060708090
100
µElution plate 3 step SOLAµ plate 3 stepCompetitor Sµ plate 3 step
Competitor: Polymeric sorbent
©2015 Waters Corporation 16
All Polymeric SPE Sorbents are NOT Created Equal
0
20
40
60
80
100
120
Oasis HLB Competitor S RP Competitor SX RP Competitor P RP
AZT
7 - Hydroxycoumarin
Phenacetin
Betamethasone
Protriptyline
Alprazolam
Methoxyverapamil
Terfenadine
Oasis AVG Recovery: 100% Oasis AVG RSD: 1%
Lower recoveries and higher variability with other sorbents
Comparative separations may not be representative of all applications.
©2015 Waters Corporation 17
History – Making SPE Simple
Mixed-mode ion exchangers
– Simplify the complex sample preparation methods to generic
procedures
– Simplified mixed mode ion exchange sample prep with 2 protocols x
4 sorbents
Will these work with a simplified protocol?
(no condition and equilibration)
©2015 Waters Corporation 18
Yes… 720005290EN
©2015 Waters Corporation 19
Moving Towards Easier Sample Preparation Techniques - Summary
Oasis HLB Simplified Protocol
Is it possible to enhance and improve the
performance of this simplified protocol?
©2015 Waters Corporation 20
Agenda
The importance of sample preparation and industry trends
Simplifying Sample Preparation
Introducing Oasis PRiME HLB
©2015 Waters Corporation 21
Introducing Oasis PRiME HLB
©2015 Waters Corporation 22
What is Oasis PRiME HLB?
PROCESS
ROBUSTNESS
improvements in…
MATRIX EFFECTS
EASE of USE
We’ll come back to this after we explain how…
What’s in a name (what does PRiME mean)?
©2015 Waters Corporation 23
Oasis PRiME HLB in 3 Words
SIMPLER
FASTER
CLEANER
©2015 Waters Corporation 24
Oasis PRiME HLB – What is it?
A reversed-phase solid phase extraction device
– PATENT PENDING
Designed to simplify solid phase extraction
– SIMPLER
o Easy protocols that result in cleaner samples
o No condition and equilibration steps are required
o Easy to implement into laboratory workflows without SPE
expertise
©2015 Waters Corporation 25
SIMPLER – In 3 Steps
3 Step Protocol – no SPE expertise required
Load:
Pre-Treated Sample
Wash:
5% MeOH
Elute:
90/10
Acetonitrile/MeOH
Does it work?
Wash and elution steps can be adjusted
if desired
©2015 Waters Corporation 26
3-Step Protocol Example: Test Analyte Properties
Name pKa Log P Comments
1B Azidothymidine (AZT) 9.68 0.05 Antiretroviral drug for HIV/AIDS
2B 7-Hydroxycoumarin 7.8 1.6 Gradient in sunscreen, absorb UV
3A Phenacetin 2.2 1.6 Pain, fever reducer
4N Betamethasone -- 1.1 Anti-inflammatory and
immunosuppressive
5B Protriptyline 8.2 4.4 Antidepressant
6A Alprazolam 2.4 4.9 Panic and anxiety disorders
7B Amitriptyline 9.7 4.8 Antidepressant
8A Naproxen 4.2 3.2 Pain, fever reducer
9B Propranolol 9.5 2.5 Hypertension
Drug Panel Mixture: Highly variable hydrophobicities, wide pKa
range and Log Ps
©2015 Waters Corporation 27
1 2
3
4 7
8
9
3-Step Protocol Example: Chromatogram
1. AZT (Azidothymidine)
2. Propranolol
3. 7-Hydroxycoumarin
4. Phenacetin
5. Protriptyline
6. Amitriptyline
7. Betamethasone
8. Alprazolam
9. Naproxen
5 6
©2015 Waters Corporation 28
3-Step Protocol Example: Recovery and Matrix Effects
-20
0
20
40
60
80
100
120
Luckycat 1cc plasma 500-500 Luckycat 1cc MERecovery Matrix Effects
3-Step Protocol CONCLUSION HIGH analyte recoveries (>80%) and
LOW (<15%) matrix effects
©2015 Waters Corporation 29
3-Step Protocol Example: Excellent Reproducibility
0
20
40
60
80
100
120
Batch #1 Batch #2 Batch #3 Batch #4 Batch #5
3-Step Protocol CONCLUSION Reproducible recoveries for all acidic, basic and neutral
compounds batch to batch, with an average recovery of 87%
©2015 Waters Corporation 30
SIMPLER – In 2 Steps
2 Step Protocol – Ideal for high organic samples, like meat or
milk extracts
Load – Collect
Matrix Interferences
Retained
Rinse – Collect
Analytes Pass Through
Unretained
Does it work?
©2015 Waters Corporation 31
Recovery of Multi-residue Veterinary from Milk (60 compounds in 9 drug classes)
One single method replaces 9 separate methods!!! Excellent recoveries ranging from 70% to 120% with precision (RSD) < 20% (n=5) for all compounds (Average recovery 91%, %RSD @ 6 (n=5)) Recovery values are a subject to the initial milk extraction efficiency
0
20
40
60
80
100
120
140
Cim
ate
rol
Cle
nbute
rol
Racto
pam
ine
Salb
uta
mol
Terb
uta
line
Tulo
bute
rol
Zilpate
rol
Clindam
ycin
Ery
thro
mycin
kitasam
ycin
Lin
com
ycin
Spiram
ycin
Tilm
icosin
Tylo
sin
Sulfachlo
rpyridazin
e
Sulfaclo
zin
e
Sulfadim
eth
oxin
e
Sulfaguanid
ine
Sulfam
era
zin
e
sulfam
ete
r
Sulfam
eth
azin
e
sulfam
eth
izole
Sulfam
eth
oxypyridazi…
sulfanilaceta
mid
e
sulfaphenazole
Sulfapyridin
e
sulfis
om
idin
e
Trim
eth
oprim
Cin
oxacin
Cip
rofloxacin
Danofloxacin
Diflo
xacin
Enoxacin
Enro
floxacin
Flu
mequin
e
Lom
efloxacin
Marb
ofloxacin
Nalidix
ic a
cid
Norf
loxacin
Ofloxacin
Orb
iflo
xacin
Oxolinic
acid
Pefloxacin
Sara
floxacin
Chlo
ram
phenic
ol
florf
enic
ol
Thia
mphenic
ol
penic
illin V
Beta
meth
asone
Cort
isone
Dexam
eth
asone
Hydro
cort
isone
Mepre
dnis
one
Meth
ylp
rednis
olo
ne
Pre
dnis
olo
ne
Triam
cin
olo
ne
Triam
cin
olo
ne
…
Cefo
taxim
e
Ceft
iofu
r
cephapirin
©2015 Waters Corporation 32
Oasis PRiME HLB – What is it?
A reversed-phase solid phase extraction device
Designed to simplify solid phase extraction
– SIMPLER
– FASTER
o Faster flows though the device
o More even flows across cartridges and plates with less plugging
o Faster overall processing with the elimination of condition and
equilibration steps combined faster flows (especially important with
cartridges)
©2015 Waters Corporation 33
FASTER - Flows
Faster, more even sample flows across cartridges and plates
with less plugging
Loading compared to Oasis HLB with 4 inch Hg, N=4
Matrix Device Format Oasis PRiME HLB Speed Increase
1:1 Diluted Plasma µElution Plate 2 - 3X Faster
1:1 Diluted Plasma 1cc / 30mg Cartridge 4X Faster
1:1 Diluted Urine 30 mg Plate 6X Faster
1:1 Diluted Urine 10 mg Plate 2X Faster
1:1 Diluted Milk 3cc / 60 mg Cartridge 1 - 2X Faster
1:1 Diluted Milk 6cc / 200 mg Cartridge 2 - 3X Faster
FASTER flows across multiple devices and sample matrices
©2015 Waters Corporation 34
FASTER – Processing Time with a 96 Well Plate
Oasis PRiME HLB
Total processing time:
9 min 30 sec
Load:
Pre-treated Sample (4 min)
Wash:
5% MeOH (3.5 min)
Elute:
90/10 Acetonitrile/MeOH
(2 min)
Competitor SPE
Total processing time:
13 min 30 sec
Condition:
MeOH (30 sec)
Equilibrate:
Water (2 min)
Load:
Pre-Treated Sample (4.5 min)
Wash:
5% MeOH (4 min)
Elute:
90/10 Acetonitrile / MeOH
(2.5 min)
SSLE
Total processing time:
28 – 33 min
Load sample, initiate (3 min)
Wait (5 – 10 min)
Add extraction solvent (2 min)
Wait (5 – 10 min)
Extract (1 min)
Evaporate (10 – 15 min)
Reconstitute (2 min )
©2015 Waters Corporation 35
Oasis PRiME HLB – What is it?
A reversed-phase solid phase extraction device
Designed to simplify solid phase extraction
– SIMPLER
– FASTER
– EVEN CLEANER
o Removes more than 95% of common matrix interferences, such as
salts, proteins and phospholipids, with the generic 3-step protocol
o Removes at least 90% more phospholipids than the generic
protocol with Oasis HLB
©2015 Waters Corporation 36
CLEANER
How does it compare to other SPE products?
©2015 Waters Corporation 37
CLEANER Eluates versus Competitors and PPT in Plasma
Oasis PRiME HLB 3-Step Protocol vs. Competitor 5-Step Protocol
CLEANER samples in fewer steps with Oasis PRiME HLB
Phospholipids Remaining in Final Eluate
Oasis PRiME HLB Competitor SX Competitor SO PPT 1:3 Plasma:ACN
Are
a C
ounts
n=5
Comparative separations may not be representative of all applications.
©2015 Waters Corporation 38
CLEANER
What can this higher level of cleanliness do for your
analytical results?
Let’s look at a typical protocol for the analysis of
veterinary drug residues in meat…
©2015 Waters Corporation 39
CLEANER - Meat Matrices (Pork) Oasis PRiME HLB vs Silica C18
Oasis PRiME HLB (60 mg) vs. Silica C18 (100 mg)
Sample Pre-Treatment
– 5 g pork
– 10 mL of 0.2% formic acid 80:20 ACN:water
– Vortex, shake 30 min, centrifuge
Solid Phase Extraction Cleanup
– Silica C18 was conditioned first with 1 mL of 0.2 % formic acid in 80:20
ACN/water
– Oasis PRiME HLB was not conditioned
Load (Pass-Through) Step
– 0.5 mL for Oasis PRiME HLB (60 mg) vs. 1.0 mL for Silica C18 (100 mg)
– Pass through and collect
– Take 200 μL of load fraction, dilute with 250 μL of 25 mM of ammonium
formate (pH 4.5) in water and 300 μL water
– Inject 5 µL
©2015 Waters Corporation 40
Silica C18
Oasis PRiME HLB
CLEANER - Pork ACN Extract Phospholipids Remaining after Pass-Through
Time1.00 2.00 3.00 4.00 5.00 6.00
%
0
1.00 2.00 3.00 4.00 5.00 6.00
%
0
LC050615_3 1: MRM of 12 Channels ES+ TIC (Phospholipid)
1.87e8
4.58
4.33
LC050615_5 1: MRM of 12 Channels ES+ TIC (Phospholipid)
1.87e8
4.33
Lipid Removal from Acetonitrile-Based Meat
Extract RESULTS: Oasis PRiME HLB removes more than 90% of hexane-extractable total lipids (determined gravimetrically). Oasis PRiME HLB successfully removes both phospholipids and fats in pass through method. The silica C18 sorbent removes only fats, NOT phospholipids. Removal of both of these components results in fewer matrix effects and less column and/or instrument contamination.
©2015 Waters Corporation 41
Oasis PRiME HLB Summary
Oasis PRiME HLB is the next generation SPE device that sets the new performance standard for routine analyses
– Best choice for samples that contain proteins, fats, and/or lipids and can be prepared by reversed-phase ‘catch-and-release’ SPE or ‘pass-through’ SPE
SIMPLER: Streamlined protocols, no condition and equilibration steps, easy to implement into laboratory workflows without SPE expertise
FASTER: Faster flows through device, more even flows across cartridges and plates with less plugging, faster overall processing
CLEANER: Reduced matrix effects, phospholipid and fat removal in the pass-through method, less column and/or instrument contamination
Load:
Pre-Treated Sample
Wash:
5% MeOH
Elute:
90/10
Acetonitrile/MeOH
Load – Collect
Matrix Interferences
Retained
Rinse – Collect
Analytes Pass Through
Unretained
2-Step Pass-Through
3-Step Catch and Release
©2015 Waters Corporation 42
Agenda
The importance of sample preparation and industry trends
Simplifying Sample Preparation
Introducing Oasis PRiME HLB
Applications and comparisons
– Endogenous Steroids in Plasma
– Synthetic Cannabinoids in Whole Blood
©2015 Waters Corporation 43
Oasis PRiME HLB vs. Protein Precipitation (PPT): Endogenous Steroids in Plasma
Modified Oasis PRiME Protocol
Oasis PRiME HLB µElution plate, N=4
Protocol
150 μL plasma (10 ng/mL)
Precipitate with 300 μL of 4:1 MeOH:ZnSO4 (89g/L)
Spin @ 3220 rcf for 10’
Dilute supernatant with 900 μL 4% H3PO4
Load pretreated sample (2 aliquots)
Wash with 2 x 200 μL of 25% MeOH
Elute with 2 x 25 μL 90:10 ACN:MeOH
Dilute with 50 μL of 25% MeOH
Inject 7.5 μL
©2015 Waters Corporation 44
Recovery Matrix Effects
Mean S.D. %RSD Mean S.D.
Cortisol 72.7% 3.1% 4.2% -19.0% 3.1%
Adione 72.5% 1.9% 2.7% -6.9% 2.2%
17-OHP 71.5% 1.9% 2.6% -4.5% 1.3%
Mean -10.1%
Oasis PRiME HLB vs. PPT: Recovery and Matrix Effects
-40%
-20%
0%
20%
40%
60%
80%
Cortisol Adione 17-OHP
Recovery
Matrix Effects
Low standard deviations (3.1% or less) demonstrate the
consistency of the extraction and cleanup seen with Oasis PRiME HLB.
Endogenous Steroids in Plasma
Absolute average matrix effect is 10%
©2015 Waters Corporation 45
Total PL Area
PPT 22152370
PRiME HLB 690579
Oasis PRiME HLB superior performance: Removal of 97% of phospholipids vs. PPT
Oasis PRiME HLB vs. PPT Phospholipid Removal
Time2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.52
2.81
PPT
Oasis PRiME HLB
PPT Protocol:
150 μL plasma (10 ng/mL)
Precipitate with 300 μL of 4:1 MeOH:ZnSO4
Spin @ 3220 rcf for 10’
Endogenous Steroids in Plasma
©2015 Waters Corporation 46
Synthetic Cannabinoids in Whole Blood – Complex Panel of Analytes
No. Compound Mol.
Formula
Cone Voltage
(V)
1˚MRM Transitions
Collision Energy
(eV)
1 AM2233 C22H23IN2O 40 459.2→98.05 34
2 RCS-4, M10 C20H21NO3 40 324.2→121.0 22
3 RCS-4, M11 C20H19NO3 36 322.2→121.0 22
4 AM 1248 C26H34N2O 56 391.4→135.1 28
5 JWH-073 4-COOH C23H19NO3 50 358.2→155.1 26
6 JWH-073 4-OH met. C23H21NO2 50 344.2→155.1 22
7 JWH-018 5-COOH C24H21NO3 46 372.2→155.1 24
8 JWH-073 3-OH met. C23H21NO2 44 344.2→155.1 26
9 JWH-018 5-OH met. C24H23NO2 40 358.2→155.1 24
10 JWH-018 4-OH met. C24H23NO2 40 358.2→155.1 24
11 JWH-015 C23H21NO 42 328.2→155.1 24
12 RCS-4 C21H23NO2 44 322.2→135.1 26
14 JWH-022 C24H21NO 50 340.2→155.1 26
13 JWH-073 C23H21NO 48 328.2→155.1 26
15 XLR-11 C21H28FNO 48 330.3→125.1 26
16 JWH-203 C21H22ClNO 46 340.2→125.0 26
17 JWH-018 C24H23NO 44 342.2→155.1 26
18 RCS-8 C25H29NO2 42 376.3→121.1 26
19 UR-144 C21H29NO 46 312.3→125.1 24
20 JWH-210 C26H27NO 48 370.2→183.1 26
21 AB 001 C24H31NO 52 350.3→135.1 30
22 AKB 48 C23H31N3O 38 366.3→135.1 22
Analyte concentrations: 100 ng/mL
©2015 Waters Corporation 47
Time
Time
0.50 1.00 1.50 2.00 2.50 3.00 3.50
4.00 4.50 5.00 5.50 6.00 6.50 7.00
%%
0
0
100
100
Chromatogram with CORTECS C18 1) AM 2223 2) RCS4, M10 3) RCS-4, M11 4) AM 1248 5) JWH-073 4-COOH met. 6) JWH-073 4-OH met. 7) JWH-018 5-COOH met. 8) JWH-073 (+/-) 3-OH
met. 9) JWH-018 5-OH met. 10)JWH-018 (+/-) 4-OH
met. 11) JWH-015 12) RCS-4 13) JWH-073 14) JWH-022 15) XLR-11 16) JWH-203 17) JWH-018 18) RCS-8 19) UR-144 20) JWH-210 21) AB 001 22) AKB 48
3
2 1
4
5
6
7
8 9, 10
13, 14
11, 12 21
15 16
17 18
20
19
22
©2015 Waters Corporation 48
Synthetic Cannabinoids in Whole Blood
RCF: relative centrifugal force
Modified Oasis PRiME Protocol
Device: Oasis PRiME HLB 30 mg plate
Replicates: 6
Protocol
– Add 100 µL spiked blood to vial
– Add 100 µL (0.1M ZnSO4/NH4CH3COO), vortex for 5 seconds
– Add 400 µL ACN, vortex for 10 seconds then centrifuge for 5 minutes
at 7000 RCF
– Take supernatant, add 1200 µL water, vortex 5 seconds to mix
– Load above solution
– Wash with 2 x 0.5 mL 25% MeOH
– Elute with 2 x 0.5 mL 90/10 ACN/MeOH
– Evaporate and reconstitute with 100 µL of 30% ACN
©2015 Waters Corporation 49
Synthetic Cannabinoids in Whole Blood - Recovery and Matrix Effects
-80
-60
-40
-20
0
20
40
60
80
100
120
3. elute with 90/10 ACN/MeOH MERecovery ME
Excellent recoveries obtained with an average RSD of 5%
Absolute average matrix effect: 17%
©2015 Waters Corporation 50
What would this synthetic cannabinoid
application look like on other SPE sorbents?
©2015 Waters Corporation 51
Synthetic Cannabinoids in Whole Blood - Procedures
Oasis PRiME HLB
– Load pre-treated sample
– Wash with 2 x 0.5 mL 25% MeOH
– Elute with 2 x 0.5 mL 90/10
ACN/MeOH
– Evaporate and reconstitute in 100 µL
30% ACN
All other SPE devices
– Condition with 1 mL MeOH
– Equilibration with 1 mL water
– Load pre-treated sample
– Wash with 2 x 0.5 mL 25% MeOH
– Elute with 2 x 0.5 mL 90/10 ACN/MeOH
– Evaporate and reconstitute in 100 µL 30% ACN
Blood sample pre-treatment
•Add 100 µL spiked blood to vial
•Add 100 µL (0.1M ZnSO4/NH4CH3COO), vortex for 5 seconds
•Add 400 µL ACN, vortex for 10 seconds then centrifuge for 5 minutes at 7000 RCF
•Take supernatant, add 1200 µL water, vortex 5 seconds to mix
SPE procedure, N=5
Required Steps
©2015 Waters Corporation 52
0
20
40
60
80
100
120
Oasis PRiME HLB 10mg 3 step Evolute ABN 10mg 5 step
Strata X RP 10mg 5 step Bond Elut Plexa RP 10mg 5 step
EVA
PLX
High Recovery and Low Variability with Oasis PRiME HLB
10mg Plate, N=5 % Recovery
Range Average % Recovery
% RSD Range
Average % RSD
Oasis PRiME HLB 90-110%
(AM2233=71%) 100 3-7 4
EVA 60-97% 85 1-17 7
STX 59-92% 80 2-27 11
PLX 46-84% 73 3-41 11
STX
Comparative separations may not be representative of all applications.
©2015 Waters Corporation 53
-100
-80
-60
-40
-20
0
20
40
60
80
100 Oasis PRiME HLB 10mg 3 step
Evolute ABN 10mg 5 step
Strata X RP 10mg 5 step
Bond Elut Plexa RP 10mg 5 step
Low Matrix Effects with Oasis PRiME HLB
Oasis PRiME HLB: Most matrix effects within 20%, maximum 43%
JWH-203 has large matrix effects with all sorbents except Oasis PRiME HLB (-11% ME)
For the last 5 compounds, large variability was observed with all competitors SPE
EVA
STX
PLX
Why is there so much ion suppression?
Comparative separations may not be representative of all applications.
©2015 Waters Corporation 54
Ion Suppression Due to Co-Elution of Phospholipid and JWH-203
Evolute ABN 10mg
Time5.40 5.60 5.80 6.00 6.20
%
0
100
5.40 5.60 5.80 6.00 6.20
%
0
100
2015_0514_05 8: MRM of 11 Channels ES+ 524.4 > 184.4 (PL 524.4)
1.60e7
100_Cann_Test_150514_8 4: MRM of 10 Channels ES+ 340.2 > 125 (JWH-203)
4.51e6
Phospholipid 524
JWH-203
JWH-203 (1-pentyl-3-(2-
chlorophenylacetyl)indole) is a
synthethic cannabinoid
JWH-203 coelutes with phospholipid
524 (Lysophosphatidylcholine) at 5.74
minutes
JWH-203
Lysophosphatidylcholine
©2015 Waters Corporation 55
Ion Suppression Due to Phospholipid Co-elution with JWH-203
Time 5.60 5.80 6.00 6.20
%
0
100
5.60 5.80 6.00 6.20
%
0
100
5.60 5.80 6.00 6.20
%
0
100
5.60 5.80 6.00 6.20
%
0
100 2.51e7
Lyso-PL (524.4 > 184.4)
Time 5.60 5.80 6.00 6.20
%
0
100
5.60 5.80 6.00 6.20
%
0
100
5.60 5.80 6.00 6.20
%
0
100
5.60 5.80 6.00 6.20
%
0
100
JWH-203 (340.2 > 125)
2.06e6
EVA
STX
PLX
Oasis PRiME HLB ME = -11%
ME = -83%
ME = -88%
ME = -75%
Comparative separations may not be representative of all applications.
©2015 Waters Corporation 56
Matrix Effects Linked to Phospholipids
Phospholipids caused substantial ion suppression for JWH-203 with all sorbents except Oasis PRiME HLB.
-100
-50
0
50
100
Oasis PRiME HLB PLX STX EVA
Phospholipids Matrix Effect
Comparative separations may not be representative of all applications.
©2015 Waters Corporation 57
High Recovery and Low Matrix Effects for JWH-203 with Oasis PRiME HLB
Highly reproducible recoveries were achieved with Oasis PRiME HLB compared to the other SPE devices. Phospholipids caused substantial ion suppression for JWH-203 with all sorbents except Oasis PRiME HLB.
-100
-50
0
50
100
Oasis PRiME HLB 10mg 3 step
Bond Elut Plexa RP 10mg 5 step
Strata X RP 10mg 5 step
Evolute ABN 10mg 5 step
Recovery Matrix Effect
PLX STX EVA
Comparative separations may not be representative of all applications.
©2015 Waters Corporation 58
Agenda
The importance of sample preparation and industry trends
Simplifying Sample Preparation
Introducing Oasis PRiME HLB
Applications and comparisons
Conclusions
©2015 Waters Corporation 59
Oasis PRiME HLB will result in…
PROCESS (no condition and equilibration steps/faster
flows/less clogging)
ROBUSTNESS (removes variability due to sample
matrix)
improvements in…
MATRIX EFFECTS (removes phospholipids, proteins, and
salts)
EASE of USE (simple methods, fewer steps, and faster
flows)
©2015 Waters Corporation 60
Questions?
©2015 Waters Corporation 61
Appendix
Certificate of Analysis
Additional Applications / Information
©2015 Waters Corporation 62
Oasis PRiME HLB Certificate of Analysis
©2015 Waters Corporation 63
Oasis PRiME HLB Certificate of Analysis
Salt Removal Test
Phospholipid Removal Test
Protein Removal Test
Low UV
©2015 Waters Corporation 64
Oasis PRiME HLB Certificate of Analysis
©2015 Waters Corporation 65
Solvent Gradient
Time Flow %A %B
0.6 70 30
1.0 0.6 50 50
2.0 0.6 45 55
2.5 0.6 5 95
3.5 0.6 5 95
3.6 0.6 70 30
4.5 0.6 70 30
Endogenous Steroids in Plasma
UPLC I-Class - FL
MPA 0.1% HCCOH in Water
MPB 0.1% HCOOH in ACN
Column HSS T3 1.8 μm; 2.1 x 50 mm
MS Xevo TQ-S
Time0.50 1.00 1.50 2.00
%
0
100
0.50 1.00 1.50 2.00
%
0
100
0.50 1.00 1.50 2.00
%
0
1000.70
1.49
1.54
Cortisol
Androstenedione
17α-OH progesterone