Transforming Sample Preparation Introducing Waters Newest Solid Phase Extraction Product

Transcription

1 Transforming Sample Preparation Introducing Waters Newest Solid Phase Extraction Product Thank you for joining us! Our Webinar will begin shortly. While you are waiting, please feel free to browse our library of Webinar content: Click below to learn more about CORTECS, our newest Solid-Core LC Column platform: Waters Corporation 1

2 Review Recorded Versions of All 5 Events Here Waters Corporation 2

3 Friendly Reminders Please use text chat functionality to submit questions during the Webinar. Upon conclusion, follow up information will be available: Recorded version of today s presentation PDF Copies of today s slides Discount Offers on Oasis PRiME HLB Sample Preparation Products Product specific information Reference materials 2015 Waters Corporation 3

4 Today s Presenter Xin Zhang Xin Zhang, Ph.D - Senior Research Chemist, Waters Corporation Today s Webinar will be presented by Xin Zhang, Senior Research Chemist at Waters Corporation. She received her Ph.D in analytical chemistry from SUNY, Buffalo in At Waters Corporation, she has worked in Consumables R&D and the Technology Advancement Department. She has been focused on evaluating different sample prep techniques such as solid phase extraction(spe), protein precipitation(ppt), solid supported liquid extraction (SSLE) as well as the Ostro Pass-through sample prepare technique. Recently, she has been working on simplified SPE procedures for the Oasis family. In addition, she also helps customers understand sample preparation by delivering educational seminars and trouble shooting Waters Corporation 4

5 Transforming Sample Preparation Xin Zhang Senior Research Chemist 2015 Waters Corporation 5

6 Transforming Sample Preparation When you think about doing sample preparation, would you rather do this? Start End 2015 Waters Corporation 6

7 Transforming Sample Preparation Or this? Start End 2015 Waters Corporation 7

8 Agenda The importance of sample preparation and industry trends Simplifying Sample Preparation Introducing Oasis PRiME HLB Applications and comparisons Conclusions 2015 Waters Corporation 8

9 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 9

10 Sample Preparation How can I turn this? Into this? 2015 Waters Corporation 10

11 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 11

12 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 12

13 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, en Danaceau et al Bioanalysis 6(6) Application Note: Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies, en 2015 Waters Corporation 13

14 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 14

15 Agenda The importance of sample preparation and industry trends Simplifying Sample Preparation 2015 Waters Corporation 15

16 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 200 µl Methanol (MeOH) 200 µl H 2 O Condition 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 16

17 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 17

18 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 18

19 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 Comparative separations may not be representative of all applications Waters Corporation 19

20 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 20

21 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 21

22 Yes EN 2015 Waters Corporation 22

23 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 23

24 Agenda The importance of sample preparation and industry trends Simplifying Sample Preparation Introducing Oasis PRiME HLB 2015 Waters Corporation 24

25 Introducing Oasis PRiME HLB 2015 Waters Corporation 25

26 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 26

27 Oasis PRiME HLB in 3 Words SIMPLER FASTER CLEANER 2015 Waters Corporation 27

28 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 28

29 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 29

30 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 30

31 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 31

32 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 32

33 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 33

34 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 34

35 Recovery of Multi-residue Veterinary from Milk (60 compounds in 9 drug classes) Cimaterol Nalidixic Oxolinic Triamcinolone 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 Trimethoprimm Cinoxacin Ciprofloxacin Danofloxacin Difloxacin Enoxacin Enrofloxacin Flumequine Lomefloxacin Marbofloxacin acid Norfloxacin Ofloxacin Orbifloxacin acid Pefloxacin Sarafloxacin Chloramphenicol florfenicol Thiamphenicol penicillin V Betamethasone Cortisone Dexamethasone Hydrocortisone Meprednisone Methylprednisolone Prednisolone 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 35

36 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 36

37 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 37

38 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 38

39 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 39

40 CLEANER How does it compare to other SPE products? 2015 Waters Corporation 40

41 CLEANER Eluates versus Competitors and PPT in Plasma Phospholipids Remaining in Final Eluate Area Co ounts 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 41

42 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 42

43 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 43

44 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 44

45 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 2015 Waters Corporation 45 Elute: 90/10 Acetonitrile/MeOH 2-Step Pass-Through Load Collect Matrix Interferences Retained Rinse Collect Analytes Pass Through Unretained

46 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 46

47 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 47

48 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 48

49 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 49

50 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 50

51 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 51

52 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 52

53 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 53

54 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 54

55 What would this synthetic cannabinoid application look like on other SPE sorbents? 2015 Waters Corporation 55

56 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 56

57 High Recovery and Low Variability with Oasis PRiME HLB Oasis PRiME HLB 10mg 3 step Strata STX X RP 10mg 5 step Evolute EVA ABN 10mg 5 step Bond PLX Elut Plexa RP 10mg 5 step mg 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 57

58 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 58

59 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-203 % 0 Time Lysophosphatidylcholine 2015 Waters Corporation 59

60 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 60 % ME = -75%

61 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 61

62 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 EVABN 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 62

63 Agenda The importance of sample preparation and industry trends Simplifying Sample Preparation Introducing Oasis PRiME HLB Applications and comparisons Conclusions 2015 Waters Corporation 63

64 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 64

65 Questions? 2015 Waters Corporation 65

66 Appendix Certificate of Analysis Additional Applications / Information 2015 Waters Corporation 66

67 Oasis PRiME HLB Certificate of Analysis 2015 Waters Corporation 67

68 Oasis PRiME HLB Certificate of Analysis Low UV Salt Removal Test Phospholipid Removal Test Protein Removal Test 2015 Waters Corporation 68

69 Oasis PRiME HLB Certificate of Analysis 2015 Waters Corporation 69

70 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 70

71 Synthetic Cannabinoids in Whole Blood - Conditions Conditions ACQUITY UPLC I-Class/MS Column ACQUITY UPLC CORTECS C18, 2.1 x 100 mm, 1.6 µm Column Temperature 30 C Sample Temperature 10 C Detection MS: Xevo TQ-D Injection Volume 5 µl/10 µl loop Run Time (Flow Rate) 8.0 min (0.6 ml/min) Mobile Phase A Water with 0.1% formic acid Mobile Phase B ACN with 0.1% formic acid Strong wash solvent 40:40:20 ACN:IPA:Water Weak wash solvent Seal wash 10% ACN 50% MeOH 2015 Waters Corporation 71

72 Thank You for Attending! Post-Event Home Page: Introductory Offer on Oasis PRiME HLB Products Buy 1 Get 1 Free * Full Webinar Recording of Today s Session w/pdf Slide Deck Compilation of TODAY S KEY Literature, Brochures etc For Questions and to Submit your Ideas for our Next Topic Please - mychemrep@waters.com *(Limit 3 Free Boxes) 2015 Waters Corporation 72