Transforming Sample Preparation Xin Zhang Senior Research Chemist
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1 Transforming Sample Preparation Xin Zhang Senior Research Chemist 2015 Waters Corporation 1
2 Transforming Sample Preparation When you think about doing sample preparation, would you rather do this? Start End 2015 Waters Corporation 2
3 Transforming Sample Preparation Or this? Start End 2015 Waters Corporation 3
4 Agenda The importance of sample preparation and industry trends Simplifying Sample Preparation Introducing Oasis PRiME HLB Applications and comparisons Conclusions 2015 Waters Corporation 4
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 5
6 Sample Preparation How can I turn this? Into this? 2015 Waters Corporation 6
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 7
8 How Good is Good Enough? Fast gradients are common and help increase throughput, but 10 parent drugs 10 metabolites 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 Waters Corporation 8
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 coelution. 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, en Danaceau et al Bioanalysis 6(6) Waters Corporation 9
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. Is it possible to The bottom chromatogram shows the earliest and latest eluting parent compounds superimposed over the phospholipid trace, illustrating coelution. 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, en solve this problem, without a lot of extra work and effort? 2015 Waters Corporation 10
11 Agenda The importance of sample preparation and industry trends Simplifying Sample Preparation 2015 Waters Corporation 11
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. 200 µl Methanol (MeOH) Well understood SPE technology Condition 5 step generic protocol 200 µl H 2 O Equilibrate 200 µl spiked dilute plasma (100 µl plasma + 100µL 4% H 3 PO 4 ) Load 200 µl 5% MeOH in H 2 O Wash 25 µl*2 MeOH Elute 2015 Waters Corporation 12
13 Oasis HLB: First Water-Wettable Sorbent Hydrophilic-Lipophilic Balanced Copolymer N O Hydrophilic monomer Lipophilic monomer Retention of polar compounds Reversed-phase retention C 18 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 13
14 Oasis uelution Protocol Comparison: Standard vs. Simplified Standard Simplified 200 µl Methanol (MeOH) Condition 200 µl H 2 O 200 µl spiked dilute plasma (100 µl plasma + 100µL 4% H 3 PO 4 ) Equilibrate Load 200 µl spiked dilute plasma (100 µl plasma + 100µL 4% H 3 PO 4 ) 200 µl 5% MeOH in H 2 O Wash 200 µl 5% MeOH in H 2 O 25 µl*2 MeOH Elute 25 µl*2 MeOH 2015 Waters Corporation 14
15 Oasis HLB vs. Competitor Recovery: Simplified Protocol µelution plate 3 step SOLAµ plate 3 step Competitor Sµ plate 3 step Competitor: Polymeric sorbent 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 2015 Waters Corporation 15
16 All Polymeric SPE Sorbents are NOT Created Equal 120 Oasis AVG Recovery: 100% Oasis AVG RSD: 1% 100 AZT Hydroxycoumarin Phenacetin 60 Betamethasone Protriptyline 40 Alprazolam Methoxyverapamil 20 0 Oasis HLB Competitor S RP Competitor SX RP Competitor P RP Terfenadine Lower recoveries and higher variability with other sorbents Comparative separations may not be representative of all applications Waters Corporation 16
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 17
18 Yes EN 2015 Waters Corporation 18
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 19
20 Agenda The importance of sample preparation and industry trends Simplifying Sample Preparation Introducing Oasis PRiME HLB 2015 Waters Corporation 20
21 Introducing Oasis PRiME HLB 2015 Waters Corporation 21
22 What is Oasis PRiME HLB? What s in a name (what does PRiME mean)? PROCESS ROBUSTNESS improvements in MATRIX EFFECTS EASE of USE We ll come back to this after we explain how 2015 Waters Corporation 22
23 Oasis PRiME HLB in 3 Words SIMPLER FASTER CLEANER 2015 Waters Corporation 23
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 24
25 SIMPLER In 3 Steps 3 Step Protocol no SPE expertise required Load: Pre-Treated Sample Wash: 5% MeOH Does it work? Wash and elution steps can be adjusted if desired Elute: 90/10 Acetonitrile/MeOH 2015 Waters Corporation 25
26 3-Step Protocol Example: Test Analyte Properties Name pk a Log P Comments 1B Azidothymidine (AZT) Antiretroviral drug for HIV/AIDS 2B 7-Hydroxycoumarin Gradient in sunscreen, absorb UV 3A Phenacetin Pain, fever reducer 4N Betamethasone Anti-inflammatory and immunosuppressive 5B Protriptyline Antidepressant 6A Alprazolam Panic and anxiety disorders 7B Amitriptyline Antidepressant 8A Naproxen Pain, fever reducer 9B Propranolol Hypertension Drug Panel Mixture: Highly variable hydrophobicities, wide pka range and Log Ps 2015 Waters Corporation 26
27 3-Step Protocol Example: Chromatogram AZT (Azidothymidine) 2. Propranolol 3. 7-Hydroxycoumarin 4. Phenacetin 5. Protriptyline 6. Amitriptyline 7. Betamethasone 8. Alprazolam 9. Naproxen Waters Corporation 27
28 3-Step Protocol Example: Recovery and Matrix Effects Recovery Luckycat 1cc plasma Matrix Effects Luckycat 1cc ME Step Protocol CONCLUSION HIGH analyte recoveries (>80%) and LOW (<15%) matrix effects 2015 Waters Corporation 28
29 3-Step Protocol Example: Excellent Reproducibility 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 29
30 SIMPLER In 2 Steps 2 Step Protocol Ideal for high organic samples, like meat or milk extracts Load Collect Matrix Interferences Retained Does it work? Rinse Collect Analytes Pass Through Unretained 2015 Waters Corporation 30
31 Recovery of Multi-residue Veterinary from Milk (60 compounds in 9 drug classes) Cimaterol Clenbuterol Ractopamine Salbutamol Terbutaline Tulobuterol Zilpaterol Clindamycin Erythromycin kitasamycin Lincomycin Spiramycin Tilmicosin Tylosin Sulfachlorpyridazine Sulfaclozine Sulfadimethoxine Sulfaguanidine Sulfamerazine sulfameter Sulfamethazine sulfamethizole Sulfamethoxypyridazi sulfanilacetamide sulfaphenazole Sulfapyridine sulfisomidine Trimethoprim Cinoxacin Ciprofloxacin Danofloxacin Difloxacin Enoxacin Enrofloxacin Flumequine Lomefloxacin Marbofloxacin Nalidixic acid Norfloxacin Ofloxacin Orbifloxacin Oxolinic acid Pefloxacin Sarafloxacin Chloramphenicol florfenicol Thiamphenicol penicillin V Betamethasone Cortisone Dexamethasone Hydrocortisone Meprednisone Methylprednisolone Prednisolone Triamcinolone Triamcinolone Cefotaxime Ceftiofur cephapirin 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%, 6 (n=5)) Recovery values are a subject to the initial milk extraction efficiency 2015 Waters Corporation 31
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 32
33 FASTER - Flows Faster, more even sample flows across cartridges and plates with less plugging 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 Loading compared to Oasis HLB with 4 inch Hg, N=4 FASTER flows across multiple devices and sample matrices 2015 Waters Corporation 33
34 FASTER Processing Time with a 96 Well Plate Oasis PRiME HLB Total processing time: 9 min 30 sec Competitor SPE Total processing time: 13 min 30 sec SSLE Total processing time: min Load: Pre-treated Sample (4 min) Wash: 5% MeOH (3.5 min) Elute: 90/10 Acetonitrile/MeOH (2 min) 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) 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 34
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 35
36 CLEANER How does it compare to other SPE products? 2015 Waters Corporation 36
37 CLEANER Eluates versus Competitors and PPT in Plasma Phospholipids Remaining in Final Eluate Area Counts n=5 Oasis PRiME HLB Competitor SX Competitor SO PPT 1:3 Plasma:ACN Oasis PRiME HLB 3-Step Protocol vs. Competitor 5-Step Protocol CLEANER samples in fewer steps with Oasis PRiME HLB Comparative separations may not be representative of all applications Waters Corporation 37
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 38
39 CLEANER - Meat Matrices (Pork) Oasis PRiME HLB vs Silica C18 Oasis PRiME HLB (60 mg) vs. Silica C 18 (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 C 18 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 C 18 (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 39
40 CLEANER - Pork ACN Extract Phospholipids Remaining after Pass-Through LC050615_3 1: MRM of 12 Channels ES TIC (Phospholipid) 1.87e Lipid Removal from Acetonitrile-Based Meat Extract RESULTS: % Silica C18 Oasis PRiME HLB removes more than 90% of hexaneextractable total lipids (determined gravimetrically) LC050615_5 1: MRM of 12 Channels ES+ TIC (Phospholipid) 1.87e8 % Oasis PRiME HLB Oasis PRiME HLB successfully removes both phospholipids and fats in pass through method. The silica C 18 sorbent removes only fats, NOT phospholipids. Removal of both of these components results in fewer matrix effects and less column and/or instrument contamination Time 2015 Waters Corporation 40
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 3-Step Catch and Release Load: Pre-Treated Sample Wash: 5% MeOH Elute: 90/10 Acetonitrile/MeOH 2-Step Pass-Through Load Collect Matrix Interferences Retained Rinse Collect Analytes Pass Through Unretained 2015 Waters Corporation 41
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 42
43 Oasis PRiME HLB vs. Protein Precipitation (PPT): Endogenous Steroids in Plasma Oasis PRiME HLB µelution plate, N=4 Protocol 150 μl plasma (10 ng/ml) Precipitate with 300 μl of 4:1 MeOH:ZnSO 4 (89g/L) 3220 rcf for 10 Dilute supernatant with 900 μl 4% H 3 PO 4 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 Modified Oasis PRiME Protocol 2015 Waters Corporation 43
44 Oasis PRiME HLB vs. PPT: Recovery and Matrix Effects 80% Endogenous Steroids in Plasma 60% 40% 20% 0% -20% -40% Recovery Matrix Effects Cortisol Adione 17-OHP 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% Absolute average matrix effect is 10% Low standard deviations (3.1% or less) demonstrate the consistency of the extraction and cleanup seen with Oasis PRiME HLB Waters Corporation 44
45 Oasis PRiME HLB vs. PPT Phospholipid Removal 100 Endogenous Steroids in Plasma % Oasis PRiME HLB PPT Protocol: 150 μl plasma (10 ng/ml) Precipitate with 300 μl of 4:1 MeOH:ZnSO rcf for % PPT Total PL Area PPT PRiME HLB Oasis PRiME HLB superior performance: Removal of 97% of phospholipids vs. PPT Time 2015 Waters Corporation 45
46 Calibration Linearity and Quality Control Results from Extracted Plasma Samples N=6 Accuracy (ng/ml) Androstenedione (R 2 =0.990, ) 17α-OH progesterone (R 2 =0.993, ) Cortisol (R2=0.996, 1-500) QC Level (ng/ml) Mean S.D. %CV Mean S.D. %CV QC Level (ng/ml) Mean S.D. %CV % 5.4% 5.7% 93.7% 6.1% 6.5% % 4.9% 5.4% % 3.4% 3.6% 92.3% 4.7% 5.6% % 2.9% 3.0% % 5.3% 5.5% 93.7% 6.1% 6.5% % 5.7% 6.0% Mean 94.9% 92.6% Mean 94.0% Linear responses over the entire calibration range R All accuracies and %CVs are within 10% 2015 Waters Corporation 46
47 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 RCS-4, M10 C20H21NO RCS-4, M11 C20H19NO AM 1248 C26H34N2O JWH COOH C23H19NO JWH OH met. C23H21NO JWH COOH C24H21NO JWH OH met. C23H21NO JWH OH met. C24H23NO JWH OH met. C24H23NO JWH-015 C23H21NO RCS-4 C21H23NO JWH-022 C24H21NO JWH-073 C23H21NO XLR-11 C21H28FNO JWH-203 C21H22ClNO JWH-018 C24H23NO RCS-8 C25H29NO UR-144 C21H29NO JWH-210 C26H27NO AB 001 C24H31NO AKB 48 C23H31N3O Analyte concentrations: 100 ng/ml 2015 Waters Corporation 47
48 Chromatogram with CORTECS C , 10 % % 11, , Time 1) AM ) RCS4, M10 3) RCS-4, M11 4) AM ) JWH COOH met. 6) JWH OH met. 7) JWH COOH met. 8) JWH-073 (+/-) 3-OH met. 9) JWH OH met. 10)JWH-018 (+/-) 4-OH met. 11) JWH ) RCS-4 13) JWH ) JWH ) XLR-11 16) JWH ) JWH ) RCS-8 19) UR ) JWH ) AB ) AKB Time 2015 Waters Corporation 48
49 Synthetic Cannabinoids in Whole Blood Device: Oasis PRiME HLB 30 mg plate Replicates: 6 Protocol Add 100 µl spiked blood to vial Add 100 µl (0.1M ZnSO 4 /NH 4 CH 3 COO), 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 Modified Oasis PRiME Protocol RCF: relative centrifugal force 2015 Waters Corporation 49
50 Synthetic Cannabinoids in Whole Blood - Recovery and Matrix Effects Recovery elute with 90/10 ACN/MeOH ME ME Excellent recoveries obtained with an average RSD of 5% Absolute average matrix effect: 17% 2015 Waters Corporation 50
51 What would this synthetic cannabinoid application look like on other SPE sorbents? 2015 Waters Corporation 51
52 Synthetic Cannabinoids in Whole Blood - Procedures Blood sample pre-treatment Add 100 µl spiked blood to vial Add 100 µl (0.1M ZnSO 4 /NH 4 CH 3 COO), 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 Oasis PRiME HLB All other SPE devices Required Steps 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 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 2015 Waters Corporation 52
53 High Recovery and Low Variability with Oasis PRiME HLB Oasis PRiME HLB 10mg 3 step Evolute EVA ABN 10mg 5 step Strata STX X RP 10mg 5 step Bond PLX Elut Plexa RP 10mg 5 step 10mg Plate, N=5 Oasis PRiME HLB % Recovery Range % (AM2233=71%) Average % Recovery % RSD Range Average % RSD EVA 60-97% STX 59-92% PLX 46-84% Comparative separations may not be representative of all applications Waters Corporation 53
54 Low Matrix Effects with Oasis PRiME HLB Oasis PRiME HLB 10mg 3 step Evolute EVA ABN 10mg 5 step Strata STX X RP 10mg 5 step Bond PLX Elut Plexa RP 10mg 5 step Why is there so much ion suppression? Oasis PRiME HLB: Most matrix effects within 20%, maximum 43% For the last 5 compounds, large variability was observed with all competitors SPE JWH-203 has large matrix effects with all sorbents except Oasis PRiME HLB (-11% ME) Comparative separations may not be representative of all applications Waters Corporation 54
55 Ion Suppression Due to Co-Elution of Phospholipid and JWH-203 Evolute ABN 10mg 2015_0514_ : MRM of 11 Channels ES > (PL 524.4) 1.60e7 JWH-203 (1-pentyl-3-(2- chlorophenylacetyl)indole) is a synthethic cannabinoid % Phospholipid 524 JWH-203 coelutes with phospholipid 524 (Lysophosphatidylcholine) at 5.74 minutes _Cann_Test_150514_8 4: MRM of 10 Channels ES > 125 (JWH-203) 4.51e6 JWH-203 % JWH Time Lysophosphatidylcholine 2015 Waters Corporation 55
56 Ion Suppression Due to Phospholipid Co-elution with JWH-203 Lyso-PL (524.4 > 184.4) JWH-203 (340.2 > 125) e e6 % % Oasis PRiME HLB ME = -11% % PLX % ME = -83% % STX % ME = -88% % EVA 0 Time 0 Time Comparative separations may not be representative of all applications Waters Corporation 56 % ME = -75%
57 Matrix Effects Linked to Phospholipids Phospholipids Matrix Effect Oasis PRiME HLB PLX STX EVA Phospholipids caused substantial ion suppression for JWH-203 with all sorbents except Oasis PRiME HLB. Comparative separations may not be representative of all applications Waters Corporation 57
58 High Recovery and Low Matrix Effects for JWH-203 with Oasis PRiME HLB Recovery Matrix Effect Oasis PRiME HLB 10mg 3 step Bond Elut PLX Plexa Strata X STX RP 10mg Evolute EVA ABN RP 10mg 5 step 5 step 10mg 5 step 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. Comparative separations may not be representative of all applications Waters Corporation 58
59 Agenda The importance of sample preparation and industry trends Simplifying Sample Preparation Introducing Oasis PRiME HLB Applications and comparisons Conclusions 2015 Waters Corporation 59
60 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
61 Questions? 2015 Waters Corporation 61
62 Appendix Certificate of Analysis Additional Applications / Information 2015 Waters Corporation 62
63 Oasis PRiME HLB Certificate of Analysis 2015 Waters Corporation 63
64 Oasis PRiME HLB Certificate of Analysis Low UV Salt Removal Test Phospholipid Removal Test Protein Removal Test 2015 Waters Corporation 64
65 Oasis PRiME HLB Certificate of Analysis 2015 Waters Corporation 65
66 Endogenous Steroids in Plasma UPLC MPA MPB I-Class - FL 0.1% HCCOH in Water 0.1% HCOOH in ACN 100 % 0.70 Cortisol Column HSS T3 1.8 μm; 2.1 x 50 mm MS Xevo TQ-S Solvent Gradient Time Flow %A %B % % 0 Androstenedione α-OH progesterone 1.54 Time Waters Corporation 66
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