What is the Current Best Method for Multiclass, Multiresidue Analysis of Veterinary Drug Residues in Animal Tissues?

Transcription

1 What is the Current Best Method for Multiclass, Multiresidue Analysis of Veterinary Drug Residues in Animal Tissues? Steven J. Lehotay, Alan R. Lightfield, Lucía Geis Asteggiante, and Marilyn J. Schneider

2 %Violations Violation Rate on Inspector Generated Bovine Slaughter Classes 1.80% beta-lactams Aminoglycosides 1.60% Sulfas Tetracyclines Macrolides Flunixin Total No. Samples 1.40% 1.20% 1.00% 0.80% 0.60% 1498 violations 400, , , , , , % 0.20% 100,000 50, % See OIG Report:

3 Veterinary Drug Residue Analysis by FSIS 6,300 Slaughter Houses FSIS Lab St. Louis, MO Inspector Samples n 150,000/yr 0.5% of cows Shipment hrs + KIS TM or FAST 3 6 hrs FDA CVM Enforcement hrs + 7 Plate Bioassay Quantitative Bioassay 24 hrs > Tol. > 7 days + < Tol. Analytical Methods* *Confirmation or Determination by Class of Drugs Slide modified from James Withee, FSIS

4 Proposed FSIS Residue Monitoring Scheme 6,300 Slaughter Houses Analytical Confirmation and Quantification < 7 days FSIS Labs St. Louis, MO Athens, GA Alameda, CA FDA CVM Enforcement > Tol. Inspector Samples n > 150,000/yr >0.5% of cows 24 hrs Shipment hrs + Bridging Studies? years + UHPLC MS/MS* KIS TM or 3 hrs Premi? *Qualitative Screening, Identification, and Semi Quantification Quantitative Bioassay 24 hrs

5 44(+13) Antibiotics in Initial MMM(+AMGs) β Lactams (10) desacetyl cephapirin, amoxicillin, DCCD (cefitofur metabolite), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, nafcillin, dicloxacillin Sulfonamides (16) sulfamethazine, sulfathiazole, sulfachloropyridazine, sulfadoxine, sulfadimethoxine, sulfapyridine, sulfadiazine, sulfamerazine, sulfaquinoxaline, sulfaethoxypyridazine, sulfanilamide, sulfamethoxazole, sulfamethizole, sulfabromomethazine, sulfanitran, sulfamethoxypyridazine Tetracyclines (3) tetracycline, oxytetracycline, chlortetracycline Macrolides (8) erythromycin, tilmicosin, lincomycin, tylosin, clindamycin, pirlimycin, tulathromycin, gamithromycin Fluoroquinolones (7) ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, desethylene ciprofloxacin, norfloxacin Aminoglycosides (13) spectinomycin, streptomycin, dihydrostreptomycin, amikacin, kanamycin, 4 gentamicins, neomycin, apramycin, hygromycin, tobramycin

6 18 Other Drugs Added to the Target List NSAIDs (6) β+dexamethasone, flunixin, oxyphenylbutazone, phenylbutazone, prednisone β agonists (5) cimaterol, clenbuterol, ractopamine, salbutamol, zilpaterol Phenicols (3) chloramphenicol, florfenicol, florfenicol amine Other (4) melengestrol acetate, zeranol, carbadox, 2 quinoxaline carboxylic acid (marker residue for carbadox) Int Stds / QC (6) sulfamethazine 13 C 6, flunixin d 3, penicillin G d 7 atrazine, methomyl, imidacloprid 62(+13) Drugs in Initial Method(s) +6 IS/QC

7 Developed, Validated, and Transferred Method for Aminoglycosides (AMGs) in Bovine Kidney, Liver, and Muscle Mix a 2.0 g sample with 10 mm NH 4 OAc / 0.4% EDTA / 1% NaCl / 2% TCA buffer in stomacher. Centrifuge and decant supernatant. Adjust ph to 6.5 with NaOH and/or HCl solutions. Sample clean up / analyte recovery performed using disposable pipette extraction (loose 50 mg WCX sorbent in 5 ml tip) on manifold extractor apparatus. Elute the analytes using 1.0 ml of 10% formic acid in H 2 O 4 fold higher sample throughput than previous USDA FSIS method

8 UHPLC MS/MS Chromatographic Profile of 13 Aminoglycosides 2.5 min >10 fold faster than previous USDA FSIS method

9 HPLC MS/MS Chromatographic Profile of Veterinary Drugs in Tissues 20 min

10 UHPLC MS/MS Chromatographic Profile of Veterinary Drugs in Tissues 7.5 min Mobile Phase: A 95% water / 5% MeCN / 0.1% formic acid B 100% MeCN / 0.1% formic acid

11 Goal: Identify in Kidney at ½ Tolerance Levels ½ Tol. (ng/g) Analytes No. 3 β Agonists, zeranol, chloramphenicol 7 5 amoxacillin, ampicillin, cloxacillin 3 10 melengestrol acetate flunixin 1 15 carbadox, 2 quinoxaline carboxylic acid 2 25 Fluoroquinolones, penicillin G 8 50 Sulfonamides, remaining NSAIDs, Macrolides, β Lactams tilmicosin florfenicol, tylosin florfenicol amine pirlimicin oxytetracycline, tetracycline chlortetracycline, tulathromycin Fixed IS/QCs 6

12 Comparison of 6 Vet. Drug MMMs 1) Mol et al. (Rikilt The Netherlands) 2) Martos et al. (U. Guelph ON, Canada) 3) Mastovska et al. (USDA ARS Wyndmoor, PA) 4) Leepipatpiboon et al. (Chulalongkorn U., Thailand) 5) Stubbings et al. (FERA York, UK) 6) Kaufmann et al. (Switzerland) Too Tedious! All methods gave similar qualitative MS/MS screening capabilities with nearly all of the drug analytes meeting identification criteria at ½ tolerance level in kidney. Speed, cost, ease of use and ruggedness become the differentiating aspects.

13 Comparison of 5 Vet. Drug MMMs ½x, 1x, and 2x Tolerance Levels (n= 6 each) in Kidney, No I.S. Mol Martos Mastovska Leepipatpiboon Stubbings 120% 100% 80% 60% 40% 20% Avg. Recoveries 6 β Lactams 7 Fluoro Quinolones 8 Growth Promoters 7 Macrolides 14 Sulfonamides 3 Tetracyclines 0%

14 Evaluation of Incurred Samples FSIS provided 10 kidneys found in their monitoring program to contain drug residues. We analyzed the samples in blind fashion (unknown drugs and unknown levels). These were each analyzed in duplicate using the different features of Mol, Martos, and Mastovska (et al.) 3 MMMs to compare and assess their performances on real samples.

15 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Pirlimycin in Incurred Kidney (no IS) 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

16 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Sulfamethazine in Incurred Kidney (no IS) 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

17 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Beta/Dexamethasone in Incurred Kidney (no IS) 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Beta/Dexamethasone was missed by FSIS Kidney Sample

18 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Flunixin in Incurred Kidney (no IS) 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

19 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Flunixin vs. Flunixin-d3 IS in Incurred Kidney 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

20 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Penicillin G vs. PenG-d7 IS in Incurred Kidney 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid No need for long shake nor heat, so fast and cool is fine! Spikes were ok, but incurred penicillins can t tolerate acids Kidney Sample

21 Goldilocks? Goldilocks? MMM for Vet. Drugs extraction clean up 2 g tissue in a 50 ml tube add IS mix (SMZ IS; flunixin d3; PenG d7) add 10 ml of 4/1 (v/v) MeCN/water vortex briefly, shake for 5 min centrifuge for 5 min >3500 rcf supernatant mg C ml hexane sat d w/mecn; mix for 30 s, centrifuge for 5 min > 3500 rcf; aspirate hexane to waste evaporate 5 ml extract to 1 ml final vol. filter extract with the Mini UniPrep TM UHPLC MS/MS analysis Modified from: K. Mastovska, A.R. Lightfield, J. Chromatogr. A 1202 (2008)

22 Method Logistics 1 chemist was able to process 60 pre homogenized samples in an 8 hr 8 day for an overnight sequence (longest step was 1 hr to evaporate MeCN) No glassware to be cleaned afterwards Cost of materials $3/sample (using bulk C18) Waste = 10 ml hexane and 5 ml MeCN (and two 50 ml and one 15 ml PP tubes)

23 Streamlined Method Validation Needs: Trueness (Recoveries at 3 3 Levels, n=10) Precision (Repeatability & Reproducibility) Ruggedness (Multi day, Multi Analyst, etc.) Selectivity (Interferences in Blanks?) Range (Calibration and Matrix Effects) Detection Limits (LOI < ½ Tol. Tol.?) Qualitative (False Negatives/Positives) Can We Meet All Needs in Days?

24 Day 1: 1 5 Day Validation Experiment Analyst 1 in hot lab, Reagents A, 10 matrix blanks from different sources, 6 spikes at 3 levels each in 6 matrices + 4 spikes each at same levels in mixed matrices (1 in glass tubes); 5 point 5 calibration each in mixed matrix and reagent only stds; reagent blk = 0 Std 0 inj d d after high std to check for carry over Days 2 & 3 (repeated on Days 4 & 5): Analysts 2 & 3 in cooler labs repeat using Reagents B & C with different sources of matrices

25 Quantitative Method Validation A) Trueness: Recoveries of Spiked Samples B) Precision: Repeatability and Reproducibility The MMM cannot be used for enforcement actions if there is a tolerance value unless the method is bridged to the FDA approved method (or it becomes an AOAC Official Method). The method may be determinative if there is no tolerance value for the analyte, or an enforcement action is not being taken.

26 Recoveries of Tylosin ( ng/g) 100% 90% 80% 70% Avg. Repeatability = 12% RSD n = 30 each day (10 reps x 3 levels) Reproducibility = 21% RSD Recovery 60% 50% 40% 30% 20% Steve Lucia Alan Marilyn Christine n = % 0% Day 1 Day 2 Day 3 Day 4 Day 5 Average

27 Average Recoveries of the 62 Drugs No. of Drug Analytes Matrix matched stds w/o internal stds n = 150 per drug (10 reps x 3 levels x 5 days) 20 different kidneys <50 %Recovery

28 Precision in the Analysis of the 62 Drugs No. of Drug Analytes Reproducibility Repeatability n = 150 per drug (10 reps x 3 levels x 5 days) 20 different kidneys >25 %RSD Using matrix matched matched stds w/o normalization to internal stds

29 Qualitative Method Validation A) Screening: Capability to replace the 7 plate bioassay in FSIS B) Identification: Capability to replace the single class LC/MS MS confirmation methods Then, the NADA (FDA approved enforcement method) is used for both quantitative determination and qualitative confirmation of drug violations.

30 Screening Criteria and Results USDA FSIS Criteria: Presence of at least one fragment ion Signal/Noise > 3 Retention time match within ±5% 54/62 analytes met screening criteria at ½ Tol. level Chloramphenicol met screening criteria at 2x Tol. level 8 drugs to be addressed in future work: desacetyl cephapirin, florfenicol amine, clenbuterol, phenylbutazone, oxyphenylbutazone, tulathromycin, cimaterol, amoxacillin

31 Our LC MS/MS Identification Criteria 1. Retention time (t R ) is within ±4 SD of average t R and peak shape matches that of reference std 2. t R and peak shape of qual. ion(s) matches those of the quantification ion 3. 2 qual. ions 20% or 1 qual. Ion 10% of avg ion ratio from contemporaneous reference stds 4. Absence of positive findings in blanks 5. Signal > ½ Tol. calibration stds in matrix

32 Ret. Time Consistency for Identification Clenbuterol Combined, n=250 Solvent Stds, n=50 Matrix Stds, n=50 4 x Std Dev (s) Tulathromycin A? 2 0 Desacetyl cephapirin and Florfenicol amine need sorted out Avg tr (min)

33 Ion Ratios for Ampicillin in Matrix Ion Ratios in Spks, n=50 each (10 x 5 days) Ion Ratio 120% 100% 80% 60% 40% 20% 0% 1/2x Spk (2/1 ions) 1x Spk (3/1 ions) 2x Spk (3/2 ions) Ion ratio variability decreases vs. conc. 1 false 5 ng/g (2%); no false positives, n= Spk Conc. (ng/g)

34 Ion Ratio Criteria in 2002/657/EC (EU) Rel. Abundance Acceptable Diff. vs. Ref. vs.. Base Peak API MS >50% ±20% RSD >20 50% ±25% RSD >10 20% ±30% RSD 10% ±50% RSD Ref. Ratio EU Range * FSIS (1 ion) (2 ions) 70% 56% 84% 60% 80% 50% 90% 24% 18% 30% 14% 34% 4% 44% 12% 8.4% 15.6% 3% 23% >0% 33% 4% 2% 6% >0% 14% >0% 24% * 2 ion transitions needed to achieve 3 ident. points in MS/MS

35 Ion Ratios for Ciprofloxacin in Kidney Ion Ratio 160% 140% 120% 100% 80% 60% 40% 20% 0% Ion Ratios of Spks, n=30 each (10 x 3 levels) Day 1 Day 2 Day 3 Day 4 Day 5 No False Positives, n=50 (EU or FSIS) Conc., False Negatives ng/g EU FSIS 25 (n=50) 54% 2% 50 (n=50) 40% 0% 100 (n=50) 26% 0% Ions 2/1 Ions 3/1 Ions 3/2

36 Ion Ratios for Lincomycin in Kidney Ion Ratio 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% Ion Ratios of Spks, n=30 each Day 1 Day 2 Day 3 Day 4 Day 5 No False Positives, n=50 (EU or FSIS) Conc., False Negatives ng/g EU FSIS 25 (n=50) 26% 0% 50 (n=50) 8% 0% 100 (n=50) 2% 0% (10 x 3 levels) Ions 2/1 Ions 3/1 Ions 3/2

37 Ion Ratios for Cimaterol Ion Ratio Ion Ratios in Stds, n=10 each 500% 400% 300% 200% Solvent 2/1 Matrix 2/1 Solvent 3/1 Matrix 3/1 Solvent 3/2 Matrix 3/2 Quant. ion gave 78±14 %recoveries, n=150 Kidney matrix interferences <20 ng/g for ions 2 & 3 1 false positive in 50 matrix blanks 100% 0% Conc. (ng/g)

38 Summary of Identification Results 41/61 drug analytes met ident. criteria at all levels 3 analytes met ident. criteria at the 1x Tol. level: desethylene ciprofloxacin, cefazolin, florfenicol 5 analytes met ident. criteria at the 2x Tol. level: sulfaniliamide, sarafloxacin, sulfanitran, cloxacillin, melengesterol acetate 12 drugs did not meet ident. criteria at the spk levels An UHPLC MS/MS instrument upgrade would lower limits of identification through dilution of extracts.

39 Veterinary Drug Residues Conclusions The streamlined method has met validation criteria for 49/61 49/61 drugs in a 5 day 5 validation for qualitative identification screening purposes. Sample throughput is 60 samples/day by 1 chemist for UHPLC MS/MS analysis. The method is being implemented for routine monitoring of cattle (so far) by the USDA labs. Quantification is acceptable for 60% of the drugs, but enforcement still requires the NADA method. Is the new MMM just right to deserve the name Goldilocks?

40 Acknowledgments Katerina Mastovska * Christine Haines * Currently at Covance Greenfield, Indiana USDA FSIS: Terry Dutko Louis Bluhm Chilton Ng Pat McCaskey et al. US Israel Binational Agricultural Research and Development Grant US

41 + +! Tanks Berry Mulch!

42

43 Thanks very much! Contacts:

44 Outline of the Talk I. Current Situation and Proposed Changes A. Feasibility Study B. Choice of Target Drugs and Levels C. Aminoglycosides Method II. Comparison of MMMs and Optimization A. Spiked Samples B. Incurred Samples III. Validation (Quantitative and Qualitative) IV. Conclusions

45 Ion Ratio 300% 250% 200% 150% 100% Ion Ratios for Sulfapyridine Ion Ratios of Spks, n=30 each Day 1 Day 2 Day 3 Day 4 Day 5 Ion source was cleaned between Days 2 & 3 ion ratios changed for some analytes 50% 0% Ions 2/1 Ions 3/1 Ions 3/2

46 Definitions Indication = result of a screening method (i.e. presumed positive or negative) Determination = result from an analytical determinative method (e.g.( GC/PFPD, LC/UV) Identification = qualitative result from a highly selective method (e.g.( GC MS, LC MS n ) Confirmation = result from 2 or more independent analyses in agreement (ideally, one of which uses a different chemical mechanism or approach)

47 Syringeless Filters Mini-UniPrep TM (Whatman) 1) Place unfiltered sample (max. 0.5 ml) in chamber. 2) Compress filter plunger into sample chamber. Clean filtrate fills reservoir bottom up. 3) Place the Mini-UniPrep TM vial in an autosampler. aqueous samples: PVDF (polyvinylidenefluoride) filter

48 LC-MS/MS Instrumentation Waters Acquity TQD = UPLC-MS/MS system UPLC (Ultra-Performance Liquid Chromatography) Pressures up to 15,000 psi Low extra-column volumes Compatible with sub-2 μm particle columns Fast analysis with good resolution Lower solvent consumption, less waste Narrow peaks fast data acquisition needed TQD - Fast triple quadrupole MS Rapid data acquisition (5 ms dwell time) Rapid positive/negative ion mode switching (20 ms)

49 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Penicillin G in Incurred Kidney (no IS) 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

50 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Sulfadimethoxine in Incurred Kidney (no IS) 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

51 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Tilmicosin vs. SMZ-d6 IS in Incurred Kidney 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

52 Analysis of Incurred Kidney (2 g) Concentration (ng/g) Tetracyclines (Sum) in Incurred Kidney (no IS) 5 min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

53 Uncertainty (U) Limitation in the Method n = 200 extractions among 20 kidneys over 5 days (n = 5) Method Step QC Std Avg. % Recovery Avg. %RSD % of U UHPLC MS/MS Atrazine 90* Cleanup Methomyl Extraction Flunixin d Homogenization Imidacloprid * Should be 100% ( 10% bias in spks vs. stds?) 11 15% probably lost during homogenization

54 Ion Ratios for Tulathromycin A Ion Ratios in Stds, n=10 each 160% 140% Solvent 2/1 Matrix 2/1 Solvent 3/1 Matrix 3/1 Solvent 3/2 Matrix 3/2 Ion Ratio 120% 100% 80% 60% 40% 20% 0% Conc. (ng/g)

55 (UHP)LC MS(/MS) Capabilities Simultaneous identification and quantification Sensitive determination in complex matrices Wide scope (analyte, matrices) Multiclass, multiresidue methods (MMMs) Lab Based Screening Identification/Confirmation Fast, wide-scope sample preparation is the key! Slide modified from Kate Mastovska

56 Two Tiered Approach for Antibiotics Tier 1: Screening Test Performed by the USDA FSIS Inspectors in the Slaughterhouse. Currently with Microbial Inhibition Tests Tier 2: Presumptive Positive Samples Sent to the USDA FSIS Laboratory for Quantitative and Qualitative Analysis. Currently with 7 plate bioassay, but we propose to use UHPLC MS/MS at least for lab based screening and antibiotic identifications

57 β Lactams Major Classes of Antibiotics Sulfonamides Tetracyclines Penicillin G Sulfadimethoxine Tetracycline Aminoglycosides Macrolides Quinolones Gentamicin C 1 Erythromycin Enrofloxacin Currently, 219 vet. drugs (including 94 antibiotics) are on our MMM list

58 Field Based Microbial Inhibition Assays FAST Premi KIS TM Pictures by Paul Pierlott

59 FAST, Premi & KIS TM Comparison Spiked Samples of Kidney (Liquid) Threshold concentration (µg/ml) Antibiotic FAST Premi KIS TM U.S. tolerance (µg/g) Penicillin G SDMX OTC Tylosin Danofloxacin >6 0.2 (L) Streptomycin Neomycin Spectinomycin M.J. Schneider, S.J. Lehotay, Anal. Bioanal. Chem. 390 (2008)

60 Comparison of 6 Vet. Drug MMMs 200% vs. SMZ d6 IS 180% 160% 140% 120% 100% 80% 60% 40% 20% Mol Martos ARS Thai Stubbings 0% beta lactam + Cephalosporin Fluoroquinolon Growth promotors Macrolides + Lincosamides Sulfas Tetracycline

61 Old School Veterinary Drug Analysis Single analyte residue methods Single class residue methods Selective multi class residue methods Slide by Kate Mastovska

62 New School Veterinary Drug Analysis Multiclass, Multiresidue Methods (MMMs) (UHP)LC MS(/MS) Slide modified from Kate Mastovska

63 Analysis of Incurred Kidney (2 g) 250 Sulfamethazine vs. SMZ-d6 IS in Incurred Kidney IS Concentration (ng/g) min shake; w/o acid 5 min shake; w/ acid 30 min shake; w/o acid 30 min shake w/ acid 60 min heat; w/o acid 60 min heat; w/ acid Kidney Sample

64 7 Plate Bioassay (Lab Screen) Pictures from FSIS

65 Microbial Inhibition Assays Relatively inexpensive and easy 24 hr sample turnaround time Not sensitive for all antibiotic classes Cannot distinguish particular analytes or mixtures Prone to unidentifiable microbial inhibitions ( 3%)( 3%) >60% of of 7 plate samples in in FSIS are positives and need to to be be confirmed by by chemical based (LC MS/MS) methods anyway Slide modified from Kate Mastovska

66 Drug Residues Identified in 235 Kidney (Exudate) Samples Antibiotics with US tolerance Number Dihydrostreptomycin 6 14,000 ng/ml 7 Streptomycin 75 ng/ml 1 DCCD 2 28 ng/ml 10 Florfenicol amine 33 ng/ml 1 Oxytetracycline 3 57 ng/ml 13 Penicillin G 2 3 ng/ml 3 Pirlimycin 2 29 ng/ml 5 Sulfamethazine ng/ml 2

67 Feasibility Study: Vet. Drug Residues in Bovine Serum and Kidney (Exudate) Analysis of 121 drug residues, including 65 antibiotics Two sample preparation and LC MS/MS methods 1) aminoglycosides (ion pairing LC) 2) MMM for veterinary drugs (reversed phase LC) Screened / analyzed 235 serum and kidney (exudate) culled dairy cow samples from a slaughterhouse M.J. Schneider, K. Mastovska, S.J. Lehotay, A.R. Lightfield, B. Kinsella, C.E. Shultz, Anal. Chim. Acta 637 (2009)

68 Addition of Quality Control Spikes, too 1) Imidacloprid added during sample homogenization 2) I.S. compounds added prior to extraction sulfamethazine d 6 ; flunixin d 3 ; Pen.G d 7 3) Methomyl added prior to the cleanup step 4) Atrazine added to final extracts RSD overall = (RSD 12 + RSD 22 + RSD 32 + RSD 42 )

69 And Then There Were Three Leepipatpiboon et al. TCA & Precipitation! Stubbings et al. Use of PSA Lowered Recoveries Kaufmann et al. WAY too Long and Intensive M M M Mol et al. 30 min Shake w/ Acid Martos et al. 60 min Heat w/ Acid Addition Mastovska et al. 5 min Shake w/o Acid

70 Drug Residues Found by LC MS/MS in Analysis of 235 Culled Dairy Cows Antibiotics no US tolerance No. of Cows Gentamicin (serum) 8 ng/ml 1 Kanamycin (serum) 1 ng/ml 1 Lincomycin (kidney) 4 ng/ml 1 Other vet drugs in kidney (exudate) Flunixin, >300 ( 1,000) ng/ml 23 (17) Beta/dexamethasone, ng/ml 7 Prednisone, <10 ng/ml 3 Drug mixtures (not counting flunixin) 12