Oasis PRiME HLB - Introducing A New Sorbent for the Sample Cleanup of Food Matrices

Transcription

1 Oasis PRiME HLB - Introducing A New Sorbent for the Sample Cleanup of Food Matrices 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 Oasis PRiME HLB Waters Corporation 1

2 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 Product specific information / Reference materials Promotional Offers Oasis PRiME Buy One Get One 30% Off All Other Oasis Products 2015 Waters Corporation 2

3 Today s Presenter Jeremy Shia, Ph.D. Marketing Manager Waters Corporation, Milford, MA Jeremy is currently the Food & Environment marketing manager at Consumables Business Unit of Water Corporation. Previously, Jeremy was a Senior Applications Chemist in our Chemistry Applications Technology group responsible for sample prep development for food testing and food safety. He has published multiple application notes using the QuEChERS method. He has worked in a variety of areas ranging from carbohydrate and vitamin analysis, to multi-residue analysis of veterinary drugs, pesticides and mycotoxins in various food matrices Waters Corporation 3

4 Oasis PRiME HLB - Introducing A New Sorbent for the Sample Cleanup of Food Matrices Jeremy Shia, Marketing Manager Meet The Experts - Webinar November 19, Waters Corporation 4

5 Agenda Why Food Safety Testing is Important Considerations for sample preparation Oasis PRiME HLB Introduction Application of Oasis PRiME HLB for Cleanup of Meat and Milk Extracts Prior to Multiresidue Veterinary Drug LC-MS/MS Analysis Summary 2015 Waters Corporation 5

6 Drivers for Food Safety Testing Regulations Surveillance, national monitoring programs, import/export Economic Brand differentiation/marketing Increased consumer prosperity, greater choice, greater competition Green lobby organic & sustainability Public Scrutiny / Media Coverage Brand protection Food scares BSE, dioxins, antibiotics, mycotoxins Adulteration/fraud o wine, orange juice, olive oil, melamine 2015 Waters Corporation 6

7 Substances Which Pose Risk Contamination in field: Environmental contaminants dioxins, PCBs, metals Natural toxic substances mycotoxins, phycotoxins, plant toxins Pesticide residues Veterinary drug residues Contamination while processing: Processing contaminants Additives illegal dyes Packaging migration Authenticity/traceability (GMO) Deliberate adulteration 2015 Waters Corporation 7

8 Scientific Challenges in Food Safety Testing Sensitivity is required to accurately identify contaminants in a wide variety sample matrices Regulated methods Emerging contaminants High throughput is a necessity Hundreds of samples Fast turnaround time Method ruggedness and reliability is essential Co-eluting endogenous materials can result in reduced assay accuracy Data quality must be maintained Better, more informed decisions 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 o o Higher, more consistent recovery Minimize matrix effects 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 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) QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) Solid-phase extraction (SPE) Objectives: Simplest technique Fastest preparation procedure Cleanest extracts Good Enough Sample Preparation 2015 Waters Corporation 11

12 Method Requirements: Multi-residue vs. Compound-specific analysis Multi-Residue/ Multi-Class Compound or Class Specific Entire procedure (sample prep & analytical method) Generic to a diverse set of analytes Specific for one compound or class of compounds Sample Preparation Protocol Goal of Sample Cleanup Simple (one or two steps) Speed Recovery & cleanup may be compromised for a large number of analytes Multi-step Maximizing recovery & matrix cleanup Minimizing interference/ion suppression Level of Sample Cleanup Minimum/moderate Maximum Detection Techniques Tandem MS, Time-of-Flight (LC-MS and GC*-MS) Single quad MS, UV, FLR, ELS, GC* (FID or MS) *GC typically requires a higher level of sample cleanup even for multi-residue analyses 2015 Waters Corporation 12

13 Introducing Oasis PRiME HLB 2015 Waters Corporation 13

14 What is Oasis PRiME HLB? What s in a name (what does PRiME mean)? PROCESS ROBUSTNESS improvements in MATRIX EFFECTS EASE of USE 2015 Waters Corporation 14

15 Oasis PRiME HLB in 3 Words SIMPLER FASTER CLEANER 2015 Waters Corporation 15

16 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 16

17 SIMPLER 3 Step Catch and Release Protocol No SPE expertise required Reversed-phase retention for aqueous samples Suitable for compound/class specific analysis Wash and elution steps can be adjusted if desired Load: Pre-Treated Sample Wash: 5% MeOH Elute: 90/10 Acetonitrile/MeOH 2015 Waters Corporation 17

18 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 18

19 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 19

20 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 20

21 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 21

22 Method Requirements: Multi-residue vs. Compound-specific analysis Multi-Residue/ Multi-Class Compound or Class Specific Entire procedure (sample prep & analytical method) Generic to a diverse set of analytes Specific for one compound or class of compounds Sample Preparation Protocol Goal of Sample Cleanup Simple (one or two steps) Speed Recovery & cleanup may be compromised for a large number of analytes Multi-step Maximizing recovery & matrix cleanup Minimizing interference/ion suppression Level of Sample Cleanup Minimum/moderate Maximum Detection Techniques Tandem MS, Time-of-Flight (LC-MS and GC*-MS) Single quad MS, UV, FLR, ELS, GC* (FID or MS) 2015 Waters Corporation 22

23 SIMPLER 2 Step Pass-Through 2 Step Protocol Ideal for high organic (ACN) samples, like meat or milk extracts Load Collect Interferences Retained Analytes Pass Through Analytes not retained by the sorbent Suitable for multi-class compounds analysis i.e. Multi-residue veterinary drugs analysis Rinse Collect Analytes Pass Through Note: Rinse step is optional 2015 Waters Corporation 23

24 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 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%, 6 (n=5)) Recovery values are a subject to the initial milk extraction efficiency 2015 Waters Corporation 24

25 Method Requirements: Multi-residue vs. Compound-specific analysis Multi-Residue/ Multi-Class Compound or Class Specific Entire procedure (sample prep & analytical method) Generic to a diverse set of analytes Specific for one compound or class of compounds Sample Preparation Protocol Goal of Sample Cleanup Simple (one or two steps) Speed Recovery & cleanup may be compromised for a large number of analytes Multi-step Maximizing recovery & matrix cleanup Minimizing interference/ion suppression Level of Sample Cleanup Minimum/moderate Maximum Detection Techniques Tandem MS, Time-of-Flight (LC-MS and GC*-MS) Single quad MS, UV, FLR, ELS, GC* (FID or MS) *GC typically requires a higher level of sample cleanup even for multi-residue analyses 2015 Waters Corporation 25

26 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 26

27 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 27

28 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 28

29 Phospholipids - A Brief Introduction Phosphate group hydrophilic head Fatty acids hydrophobic tails Lipids Amphipathic, surfactant-like property Serve as a major structural component of most biological membranes Form lipid bilayer in cell membranes of organisms Example: phosphatidylcholine or lecithin Often found in animal and plant tissues, such as egg yolks, milk, soybeans, animal cells 2015 Waters Corporation 29

30 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 30

31 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 passthrough 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 2015 Waters Corporation 31

32 Application of Oasis PRiME HLB for Cleanup of Meat and Milk Extracts Prior to Multiresidue Veterinary Drug LC-MS/MS Analysis 2015 Waters Corporation 32

33 US DA Residue Methods for Veterinary Drug Analysis Method Analytes Sample Matrix Combined R04b β-adrenergic agonists Muscle R06 β-lactam Kidney and muscle R08b Chloramphenicol Muscle R18 Macrolide Liver, kidney, and muscle R27d Sulfonamides Liver and muscle R32 Fluoroquinolone Liver and muscle R46 Penicillin G Kidney, liver, and muscle Using Oasis PRiME HLB -Enable multi-class compounds to be analyzed using one sample prep method Waters Corporation 33

34 Challenges Creating a Single Extraction Method Compound diversity Large number of diverse group and classes Different physical and chemical properties o Polarity o pka Parent drugs and their metabolites Matrix complexity Matrix effects proteins, fats, phospholipids, salts Protein binding Low limits of detection vs. co-extracted material ion suppression Chelating - Tetracycline and similar drugs can form bonds with calcium (milk matrix) 2015 Waters Corporation 34

35 Multi-Residue Analysis - Composition of Milk and Meat Typical Cow s Milk Approximately 14 % solids o 4 % fat o % phospholipid o 4 % protein o 5 % sugar (lactose) o 85 % water Typical Pork Muscle Approximately 30 % solids o 5-20 % fat o 1-3 % phospholipid o % protein o ~70% water 2015 Waters Corporation 35

36 Veterinary Drug Classes Tetracycline Fluoroquinolone Sulfonamide Macrolide tetracycline enrofloxacin LogP -1.3, pka -2.2 sulfamethazine erythromycin LogP 2.37, pka(basic)8.3 Beta-Lactam Steroid Amphemicol Beta-adrenergic oxacillin LogP 2.05, pka 3.75 dexamethasone Chloramphemicol salbutamol LogP 0.44, pka (basic) Waters Corporation 36

37 Veterinary Drugs Extraction Methods Typical Sample Preparation Strategies Precipitation/extraction with strong buffer (McIlvaine ph 4) followed by SPE o good for tetracyclines, beta-adrenergics, polar sulfonamides, fair for fluoroquinolones, o poor recovery of most other compounds Precipitation/extraction with 3:1 acidic acetonitrile with SPE cleanup o excellent protein precipitation o poor recovery of tetracyclines, beta-adrenergics, polar sulfonamides o good recovery of most other compounds 2015 Waters Corporation 37

38 Typical %recoveries from milk Comparison of precipitation/extraction techniques Drug Class 3:1 ACN Aq Buffer 1:1 ACN* β-adrenergic <10 ~100 >80 Tetracycline <25 >70 >25 Fluoroquinolone >50 >50 >50 Macrolide >60 <35 >60 Beta-Lactam >70 <30 >70 Steroid >70 <10 >70 See also: Stolker et. al., Anal. Bioanal. Chem. 391, 2309 (2008) 2015 Waters Corporation 38

39 Why not QuEChERS for Sample Prep of Veterinary Drugs? Some classes of veterinary drugs have good recovery using QuEChERS (i.e., avermectins) However, too many have low recovery (< 50%) First step of veterinary drug extraction is similar However, no salts are added for veterinary drug analysis and pass through SPE is preferred Pesticides are generally more non-polar and less water-soluble than veterinary drugs At least 80% partition into ACN layer when DisQuE salts are added In comparison, a larger portion of veterinary drugs partition into the salt-saturated water layer 2015 Waters Corporation 39

40 Multi-Residues Veterinary Drug Analysis - Sample Extraction MILK Protein Precipitation Add 4 ml of 0.2 % formic acid ACN to 1 ml of sample TISSUE Extraction/Precipitation Add 10 ml of 0.2 % formic acid in 80:20 ACN/water to 2.5 g of sample Provides good recovery of most analytes Removes proteins sugars and salts Centrifuge Take aliquot Pass-Through SPE Cleanup Waters Application Notes Optimized Extraction and Cleanup Protocols for LC/MS/MS Multi-Residue Determination of Veterinary Drugs in Milk ( en) Optimized Extraction and Cleanup Protocols for LC-MS/MS Multi-Residue Determination of Veterinary Drugs in Edible Muscle Tissues ( en) 2015 Waters Corporation 40

41 SPE Strategy for Vet Drugs Pass-Through Cleanup* Sep-Pak C 18 Removes fats and non-polar pigments Pass-Through SPE * Cleanup Oasis PRiME HLB Removes fats, non-polar pigments and phospholipids *Also known as Chemical Filtration 2015 Waters Corporation 41

42 Oasis PRiME HLB 1 Step Pass-Through - No Rinse Step, Even More SIMPLER 1 Step Protocol Ideal for high organic (ACN) samples, like meat or milk extracts 1-Step Pass-Through Analytes not retained by the sorbent Suitable for multi-class compound analysis i.e Multi-residue veterinary drugs analysis Pass-Thru and Collect Matrix Interferences Retained 2015 Waters Corporation 42

43 Multi-Residue Veterinary Drugs in Milk and Tissue Sample pre-treatment (Milk): To 1mL of milk, add 4 ml of 0.2% formic acid (FA) in acetonitrile (ACN), mix well. Centrifuge for 5 min at rpm. An aliquot of the supernatant is taken for SPE cleanup. Sample pre-treatment (Tissue): Extract 2.5 g tissue with 10 ml of 0.2% formic acid (FA) in 80:20 ACN/Water and mix well. Centrifuge for 5 min at rpm. An aliquot of the supernatant is taken for SPE cleanup. Pass-Thru Cleanup: The vacuum was set to 1-2 psi. Approximately 0.5 ml of the supernatant was passed-through the Oasis PRiME cartridge and collected. A 0.3 ml aliquot of the pass-thru cleanup sample was taken and diluted three-fold with aqueous 10 mm ammonium formate buffer (ph 4.5) prior to UPLC- MS/MS analysis. * Typically, an exact portion of the pass-thru fraction is evaporated or simply diluted with mobile phase, depending on the instrumental conditions 2015 Waters Corporation 43

44 UPLC Conditions LC system: ACQUITY UPLC I-Class Column: ACQUITY UPLC CSH C18, 1.7µm, 100 mm x 2.1 mm (ID) Mobile phase: A: 0.1% formic in water B: 0.1% formic acid in acetonitrile Injection volume: 5 μl Injection mode: partial loop injection Column temperature 30 C Weak Needle Wash: 10:90 acetonitrile:water (600 μl) Strong Needle Wash: 50:30:40 water:acetonitrile:ipa (200 μl) Seal wash: 10:90 acetonitrile: water Gradient Time (min) Flow (ml/min) % A % B Curve Initial Initial Waters Corporation 44

45 MS Conditions Mass Spectrometer: Waters Xevo TQ-S Positive Electrospray (except negative for chloramphenicol) Source Temperature: 150 C Desolvation Temperature: 500 C Desolvation Gas Flow: 1000 L/Hr Cone Gas Flow: 30 L/Hr Collision Gas Flow: 0.15 ml/min Data Management: MassLynx v Waters Corporation 45

46 MRM Transitions Compounds MRM Cone(V) Collision(eV ) Spike Level (low,high) µg/kg Calibration Range µg/kg Corr (R 2 ) RT Compounds MRM Cone(V) Collision(eV ) Spike Level (low,high) µg/kg Calibration Range µg/kg Corr (R 2 ) RT Amoxicillin 366.2> , Oxacillin 402.2> , > > Carbadox 263.0> , Oxytetracycline 461.2> , > > Ceftiofur 524.3> , Penicillin 335.2> , > > Chloramphenicol 321.0> , Phenylbutazone 309.4> , > > Chlortetracycline 479.3> , Ractopamine , > > Ciprofloxacin 332.1> , Salbutamol 240.2> , > > Cortisol 363.2> , Sulfamerazine 265.0> , > > Dexamethasone 393.2> , Sulfamethazine 279.1> , > > Enrofloxacin 360.4> , Sulfanilamide 156.0> , > > Erythromycin 734.4> , Tetracycline 445.3> , > Lincomycin 407.2> , Tylosin 916.5> , > > Lomefoxacin 352.1> , > Waters Corporation 46

47 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 Recovery of Multi-residue Veterinary from Milk Multi-residue veterinary drugs in milk including 60 compounds in 9 drug classes compounds Excellent recoveries were obtained in the range of 70% to 120% with precision (RSD) < 20% (n=5) for all compounds (Average recovery 91%, 6 (n=5)) 2015 Waters Corporation 47

48 Phospholipids Removal From Milk Milk sample processed by protein precipitation only Precipitated milk sample cleaned up with Oasis PRiME HLB Oasis PRiME HLB removed many more phospholipids compared to protein precipitation 2015 Waters Corporation 48

49 Phospholipid Removal From Shrimp Extract 2015 Waters Corporation 49

50 Recovery Recovery of Veterinary Drugs from Salmon Tissue (9 Classes of Drugs) Total Method Recovery Recovery from Salmon Waters Corporation 50

51 Recovery Recovery of Veterinary Drugs from Shrimp Tissue (9 Classes of Drugs) Total Method Recovery Recovery from Shrimp Waters Corporation 51

52 Recovery of Veterinary Drugs Discussion Method recovery averages ~ 80% with range from % Most of any recovery loss occurs in the initial extraction/precipitation step o Not all classes of compounds are extracted equally Penicillins > 70% Sulfonamides > 85% Fluroquinolones> 75% Tetracyclines > 40% What recovery losses are caused by the pass-thru cleanup? 2015 Waters Corporation 52

53 Recovery Oasis PRiME HLB Pass-Thru Cleanup Recovery Salmon matrix blank extracts were spiked with the veterinary drugs and then cleaned up by passing through the Oasis PRiME HLB cartridge. This experiment eliminates the recovery loss contribution from the initial protein precipitation/extraction. This salmon data presented below shows the pass-thru cleanup efficiency versus the total method recovery. Shrimp data were similar. 140 Recovery from Salmon Method Recovery Pass-Thru Recovery Waters Corporation 53

54 Options for Compounds Like Tetracycline Option A Based on the method validation result, build in concentration factor when considering the reporting levels. Perform confirmation test if needed. Option B Exclude tetracycline from the list of this screening method. Analyze tetracyclines using individual analytical method o Tetracyclines are strong chlelating agents EDTA or oxalic acid is added to the McIlvaine buffer to sequester calcium o Follow by Oasis MAX cleanup Option C Extract sample again using the optimized method for tetracycline, and combine the extracts together. After adjusting the solvent by adding ACN, then pass through Oasis PRiME HLB Waters Corporation 54

55 Summary Worldwide regulatory requirements are expanding to include more compounds 400+ pesticides and 100+ veterinary drugs Need for good multi-residue screening methods are required to meet these regulations Need maximum number of compounds in a minimum number of methods for multi-residue screening PRiME HLB operated as chemical filter. Interference such as fatty matrix and phospholipids are retained by the sorbent, and analytes are allowed to be passed through. Oasis PRiME HLB effectively removes majority of fatty matrix and phospholipids from milk and tissues, and gives the cleanest possible sample extracts for multi-residue veterinary drug analysis Waters Corporation 55

56 Questions? 2015 Waters Corporation 56

57 Thank You for Attending! Post-Event Home Page: Promotional Offers Oasis PRiME Buy One Get One 30% Off All Other Oasis Products Full Webinar Recording w/pdf Slide Deck Compilation of KEY Literature, Brochures etc For Questions and to Submit your Ideas for our Next Topic Please - mychemrep@waters.com 2015 Waters Corporation 57