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Multi-class, multi-residue method (MMM) for more veterinary drug residues in animal tissues
Lucía Geis-Asteggiante, Steven J. Lehotay, and Alan R. Lightfield
Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture above others of a similar nature not mentioned
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Research GoalsResearch Goals1. Extend our existing analytical multiresidue extraction
method from 62 to 127 drug analytes.
2. Evaluate extraction method ruggedness by testing various clean-up techniques to reduce matrix effects (MEs) without sacrificing the integrity of results.
3. Validate an analytical method based on liquid chromatography tandem mass spectrometry toscreen, identify and quantify chemical residues of interest in foods.
• Increase sample throughput and reduce costs for the USDA-Food Safety and Inspection Services (FSIS).
• Reduce the hold time of suspected carcasses.
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Group 1Veterinary Drug Residues of InterestVeterinary Drug Residues of Interest
MacrolidesMacrolides (8)(8) erythromycin A, tilmicosin, lincomycin, tylosin, clindamycin, pirlimycin, tulathromycin A, gamithromycin
ββ--LactamsLactams (10)(10) desacetyl cephapirin, amoxicillin, DCCD (cefitofurmetabolite), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, nafcillin, dicloxacillin
SulfonamidesSulfonamides (16)(16) sulfamethazine, sulfathiazole, sulfachloropyridazine, sulfadoxine, sulfadimethoxine, sulfapyridine, sulfadiazine, sulfamerazine, sulfaquinoxaline, sulfaethoxypyridazine, sulfanilamide, sulfamethoxazole, sulfamethizole, sulfabromomethazine, sulfanitran, sulfamethoxypyridazine
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Group 1
Veterinary Drug Residues of InterestVeterinary Drug Residues of Interest
TetracyclinesTetracyclines (3)(3) tetracycline, oxytetracycline, chlortetracycline
ββ--AgonistsAgonists (5)(5) cimaterol, clenbuterol, ractopamine, salbutamol, zilpaterol
FluoroquinolonesFluoroquinolones (7)(7) ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, desethylene ciprofloxacin, norfloxacin
PhenicolsPhenicols (3)(3) chloramphenicol, florfenicol, florfenicol Amine
NSAIDsNSAIDs(3(3)) flunixin, phenylbutazone, oxyphenbutazone
Others Others (5)(5) beta or dexamethasone, prednisone, 2-quinoxalinecarboxilic acid, melengesterol acetate, zearalanol
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Group 2Veterinary Drug Residues of InterestVeterinary Drug Residues of Interest
AnthelminticsAnthelmintics (27)(27) abamectin, albendazole, 2-amino-albendazole sulfone, albendazole sulfone, albendazole sulfoxide, cambendazole, doramectin, emamectin benzoate, eprinomectin, fenbendazole, fenbendazole sulfone, flubendazole 2-amino-flubendazole, haloxon, ivermectin, levamisole, mebendazole, 2-amino-mebendazole, morantel, moxidectin, oxfendazole, oxibendazole, selamectin, thiabendazole, 5-hydroxythiabendazole, triclabendazole, triclabendazole sulfoxide
FlukicidesFlukicides (7)(7) bithionol, clorsulon, closantel, niclosamide, nitroxynil, oxyclozanide, rafoxanide
ThyreostatsThyreostats (6)(6) 2-thiouracil, 2-mercaptobenzimidazole, 2-mercapto-1-methylimidazole, 6-methyl-2-thiouracil, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil
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Group 2Veterinary Drug Residues of InterestVeterinary Drug Residues of Interest
TranquilizersTranquilizers (13)(13) acetopromazine, azaperone, carazolol, chlorpromazine, diclofenac, dipyrone, haloperidol,ketoprofen, promethazine, propionylpromazine, tolfenamic acid, triflupromazine, xylazine
AntiAnti--bacterialsbacterials (3)(3) bacitracin, novobiocin, virginiamycin M1
NitroimidazolesNitroimidazoles (7)(7) dimetridazole, dimetridazole-hydroxy, ipronidazole, ipronidazole hydroxy, metronidazole, metronidazolehydroxy, ronidazole
Others Others (3)(3) doxycycline, oleandamycin triacetate, orbifloxacin
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Our MMM for Veterinary DrugsOur MMM for Veterinary Drugs
add 10 mL of 4/1 (v/v) MeCN/watervortex briefly, shake for 5 mincentrifuge for 5 min >3500 rcf
2 g tissue in a 50 mL tube, add IS mix
evaporate 5 mLs extract and dilute with water to a final volume of 1 mL
add atrazinefilter extract with the syringeless PVDF filter vial
UHPLC-MS/MS analysis
supernatant + 500 mg C18 + 10 mL hexane sat’dw/MeCN; mix for 30 s, centrifuge for
5 min > 3500 rcf; aspirate hexane to waste
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UHPLC Method ParametersUHPLC Method ParametersØ Column – Acquity UPLC HSS T3, 1.7 µm, 100 x 2.1 mmØ Injection volume – 20 μL
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UHPLCUHPLC--MS/MS MS/MS Chromatographic Profile Chromatographic Profile of of VD Group 1 in Bovine MuscleVD Group 1 in Bovine Muscle
6.25min
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PostPost--Column Infusion for Column Infusion for AnthelminticsAnthelminticsSignal enhancement for selamectin using post-column infusion of 27 mM ammonium formate in MeOH-MeCN(75:25) at a rate of 50 μL/min
200 ng/g equivalent sample concentration without NH4
+
in the mobile phase.
100 ng/g equivalent sample concentration with NH4
+ added by post-column infusion.
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8.6min
UHPLCUHPLC--MS/MS MS/MS Chromatographic Profile Chromatographic Profile of of VD Group 2 in Bovine MuscleVD Group 2 in Bovine Muscle
Start ofinfusion
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Ø Check responses of
Ø Compare multi-day calibration standards
Ø Evaluate consistency in recoveries and precision of multi-day spiked samples
Ø Evaluate consistency of ion ratios (2/1, 3/1, 3/2)
Ø Assess matrix effects (MEs) and study possibilities to overcome them
Ruggedness EvaluationRuggedness Evaluationü Internal standards (IS)ü Quality control (QC)
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Ø Use an isotopically-labeled IS if possible
Ø Reduction of the amount of co-extractives by improving the extraction and clean-up process
Ø More selective chromatographic separation with the matrix
Ø External matrix-matched calibration standards
Ø Post-column infusion of the standards
Assessing and Overcoming MEsAssessing and Overcoming MEs
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Evaluation of Effectiveness Evaluation of Effectiveness for Each Cleanfor Each Clean--Up Protocol Up Protocol
Ø Measure removal of co-extracted matrix components by weight differences
Ø Measure degree of matrix effects during LC-MS/MS
Ø Measure recoveries and repeatability for each analyte
CleanClean--Up Methods TestedUp Methods TestedØ HexaneØ C18Ø Hexane + C18
Ø Z-SepØ Z-Sep + HexaneØ Z-Sep + C18Ø Z-Sep + C18 + Hexane
Ø Z-Sep+Ø Z-Sep+ + Hexane
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Effectiveness of CleanEffectiveness of Clean--UpUpin Removing Coin Removing Co--ExtractivesExtractives
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ME Profiles Before and After CleanME Profiles Before and After Clean--Up Up
Suppression is not reduced the front of the run
Z-Sep + hexane shows enhancement
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Effect of CleanEffect of Clean--Up on RecoveriesUp on Recoveries
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Effect of CleanEffect of Clean--Up on Up on RepeatabilitiesRepeatabilities
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Dilution Effects on ME Profiles Dilution Effects on ME Profiles Ø Extracts obtained after C18 + hexane clean-up were diluted by
a dilution factor (DF) of 2 and 4.
Numbers of false negatives at lowest spiking level increased vs. DF
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Design of the Design of the Validation ExperimentValidation ExperimentMatrix blanks were beef cow, dairy cow, heifer, Matrix blanks were beef cow, dairy cow, heifer, bovine and steerbovine and steer4 different sources of C4 different sources of C--18 were used18 were usedDay Day 11::ØØ Analyst Analyst 1, Reagent 1, Reagent A, 10 matrix A, 10 matrix blanks, blanks, 6 spikes at 6 spikes at
3 levels each in 6 matrices + 4 spikes each at same 3 levels each in 6 matrices + 4 spikes each at same levels in mixed levels in mixed matrices; matrices; 55--point calibration each point calibration each in mixed in mixed matrixmatrix--matched and matched and reagentreagent--only only stdsstds; ; reagent reagent blkblk = 0= 0--Std Std inj’dinj’d after high std to check for after high std to check for carrycarry--over over
Days 2, 3 & 4 (added Day 5 & 6 for VD Group 2)Days 2, 3 & 4 (added Day 5 & 6 for VD Group 2)::ØØ Analysts 2 and 3 repeat using Reagents B, C & DAnalysts 2 and 3 repeat using Reagents B, C & D
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GoalGoal:: Identify in Identify in Muscle Muscle at ½ “Tolerance” Levelsat ½ “Tolerance” Levels½ “Tol.” (ng/g) Analytes (Group 1) No.
3 ββ--Agonists, Agonists, zeranolzeranol, , chloramphenicolchloramphenicol 75 amoxacillinamoxacillin, , ampicillinampicillin, , cloxacillincloxacillin 3
10 melengestrolmelengestrol acetateacetate 112.5 flunixinflunixin 115 carbadoxcarbadox, 2, 2--quinoxaline carboxylic acidquinoxaline carboxylic acid 225 FluoroquinolonesFluoroquinolones, penicillin G, penicillin G 850 SulfonamidesSulfonamides, , remaining NSAIDs, remaining NSAIDs, MacrolidesMacrolides, , ββ--LactamsLactams 3160 tilmicosintilmicosin 1
100 florfenicol, florfenicol, tylosintylosin 2150 florfenicolflorfenicol amineamine 1250 pirlimicinpirlimicin 1500 oxytetracyclineoxytetracycline, tetracycline, tetracycline 2
1000 chlortetracycline, tulathromycinchlortetracycline, tulathromycin 2400 Fixed IS/QCs 6
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GoalGoal:: Identify in Identify in Muscle Muscle at ½ “Tolerance” Levelsat ½ “Tolerance” Levels
½ “Tol.” (ng/g) Analytes (Group 2) No.0.5 ipronidazoleipronidazole 12.5 ipronidazoleipronidazole--OHOH 15.0 anthelminticsanthelmintics, , flukicidesflukicides, tranquilizers, , tranquilizers, nitroimidazolesnitroimidazoles 25
12.5 22--mercaptobenzimidazole, mercaptobenzimidazole, oxfendazoleoxfendazole 225 anthelminticsanthelmintics, , flukicidesflukicides, , nitroimidazolesnitroimidazoles, , thiouracilthiouracil 1350 anthelminticsanthelmintics, , flukicidesflukicides, anti, anti--bacterial, bacterial, doxycyclinedoxycycline 1075 oleandomycinoleandomycin triatatetriatate 1
100 22--MMI, MMI, dipyronedipyrone, , diclofenacdiclofenac acid, acid, eprinomectineprinomectin, , selamectinselamectin 5200 thiouracilsthiouracils, , fenbendazolefenbendazole, , tolfenamictolfenamic acidacid 5250 bacitracinbacitracin 1500 novobiocinnovobiocin, , fenbendazolefenbendazole sulfonesulfone 2
400 Fixed IS/QCs 5
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QC resultsQC results• n = 90-120 extractions among 24 muscle samples
Method Step QC-StdVD
Group #
Avg. % Rec
Avg.%RSD
UHPLC-MS/MS Atrazine 1
2104102
145
ExtractionFlunixin-d3
12
8486
138
Sulfamethazine 13C612
8588
19*11
* Excludes data from day 4 as solution may have been wrongly prepared or standard may have degraded.
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1. One of the ion transitions is present in the chromatogram
2. Retention time (tR) is within ± 0.1 min of average tR and peak shape matches that of reference std
3. Signal to noise rate (S/N) > 3 for drugs that have no tolerance
4. Signal > ½ “Tolerance” calibration standards in matrix
5. Signal > ½ “Tolerance” contemporaneous fortified sample
6. No carry over or lab contamination
7. Rate of false positives ≤5% and no false negatives
Our Our UHPLCUHPLC--MS/MS MS/MS Screening Screening CriteriaCriteria
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1. Retention time (tR) is within ± 0.1 min of average tR and peak shape matches that of reference std
2. tR 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. No carry over or lab contamination
5. Signal > ½ “Tolerance” calibration standards in matrix
6. Rate of false negatives ≤ 10%
Our Our UHPLCUHPLC--MS/MS MS/MS IdentificationIdentification CriteriaCriteria
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Results for Screening and Identification Results for Screening and Identification Results at ½ “tolerance”
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Average RecoveriesAverage Recoveries
For VD Group 2 For VD Group 2 –– endcappedendcapped C18 C18 was found to be more effective was found to be more effective
for for quantitiativequantitiative resultsresults
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Average Average ReproducibilityReproducibility
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ConclusionsConclusionsØ The streamlined method has met the screening
criteria for 89% of the drugs in a 4-day validation study.
Ø For identification purposes, the method has met validation criteria for 77% of the drugs.
Ø Quantification is acceptable for 69% of the drugs, but enforcement still requires the FDA method.
Ø This screening/identification UHPLC-MS/MS method is being implemented for routine monitoring of cattle by the USDA labs.
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AcknowledgmentsAcknowledgments
Ø FSIS: Terry Dutko, John Wippler, Louis Bluhm, Chilton Ng
Ø Katerina MastovskaØ US- Israel Binational Agricultural Research and
Development FundØ University of the Republic of Uruguay
THANK YOU FOR YOUR ATTENTION!