"'Is"'Is itit EyeEye oror Pee?'Pee?' ChallengesChallenges toto SampleSample PrepPrep andand AnalysisAnalysis inin VeterinaryVeterinary Forensics"Forensics" KennethKenneth PeckPeck TexasTexas VeterinaryVeterinary MedicalMedical DiagnosticDiagnostic LaboratoryLaboratory CollegeCollege Station,Station, TXTX AnimalAnimal DrugDrug TestingTesting HighlyHighly DiverseDiverse SampleSample TypesTypes andand situationssituations –– RacingRacing SamplesSamples UseUse thethe smallestsmallest amountamount ofof aa drugdrug toto changechange performanceperformance –– LivestockLivestock ShowShow AnimalsAnimals DetectDetect drugsdrugs includingincluding growthgrowth promoterspromoters inin numerousnumerous speciesspecies withwith differentdifferent FDAFDA andand USDAUSDA requirements.requirements. FoodFood safetysafety requirementsrequirements –– PrePre--purchasepurchase drugdrug screens,screens, animalanimal poisoningspoisonings andand productproduct tampering.tampering. DrugDrug DiversityDiversity Aspirin,Aspirin, caffeine,caffeine, coughcough syrupssyrups NarcoticsNarcotics inin smallsmall dosesdoses actact asas stimulantsstimulants inin horses,horses, catscats andand bears.bears. AntibioticsAntibiotics andand foodfood safetysafety (drug(drug residues)residues) TranquilizersTranquilizers andand sedativessedatives BronchodilatorsBronchodilators GrowthGrowth promoterspromoters BetaBeta AgonistsAgonists BronchodilatorBronchodilator andand GrowthGrowth PromoterPromoter –– ClenbuterolClenbuterol –– AlbuterolAlbuterol –– TerbutalineTerbutaline –– PirbuterolPirbuterol –– ProcaterolProcaterol –– RactopamineRactopamine PayLeanPayLean (Porcine(Porcine andand Bovine)Bovine) –– ZilpaterolZilpaterol ZilMaxZilMax (Bovine(Bovine restricted)restricted) SampleSample TypeType andand TargetTarget AnalysisAnalysis HorseHorse RacingRacing –– Urine/Blood.Urine/Blood. RelativeRelative shortshort term.term. UsedUsed forfor thethe Bronchodilator/Bronchodilator/ cardiovascularcardiovascular effects.effects. HorseHorse urineurine isis veryvery unique.unique. HasHas thethe consistencyconsistency ofof warmwarm honeyhoney oror molasses.molasses. RequiresRequires largelarge porepore andand fritsfrits inin solidsolid phasephase andand MIPMIP extractions.extractions. DilutionDilution doesdoes notnot alwaysalways solvesolve thethe problemproblem SampleSample TypeType andand TargetTarget AnalysisAnalysis LivestockLivestock ShowsShows –– UrineUrine thethe routineroutine samplesample forfor bronchodilatorbronchodilator use.use. SuitableSuitable forfor solidsolid phasephase andand MIPMIP use.use. RetinaRetina harvestedharvested postpost slaughterslaughter forfor growthgrowth promoterpromoter use.use. Muscle/Liver/KidneyMuscle/Liver/Kidney LowLow drugdrug concentrations/Tissueconcentrations/Tissue manipulationmanipulation HorseHorse UrineUrine BetaBeta AgonistAgonist MIPMIP andand thethe NonNon-- IdealIdeal SampleSample Horse Urine. Very viscous/ Diluted 1:10 typically only 20% of the sample will successfully load on the column. Alternative approach. – Activate the column – Using a blunt probe (straighten paper clip) Recover/transfer the packing material. – Standard sample prep (internal standard) with ammonium acetate and adjust pH. Centrifuge sample and decant – Add MIP packing material, Allow binding by inversion – Centrifuge and decant or aspirate leaving the packing material – Transfer the packing material into a centrifugal filter. – Wash/elute with appropriate buffers/solvents using centrifugation RetinaRetina SeparateSeparate retinaretina fromfrom conjunctiva/scleraconjunctiva/sclera ofof eye.eye. HomogenizeHomogenize ProteaseProtease DigestDigest BetaBeta--GlucuronidaseGlucuronidase // SulfataseSulfatase AdditionAddition ofof acetateacetate bufferbuffer andand pHpH CentrifugationCentrifugation andand suitablesuitable forfor MIPMIP LowLow recoveryrecovery isis routinelyroutinely seen.seen. AlternativeAlternative TissueTissue SampleSample PrepPrep AfterAfter homogenationhomogenation // digestiondigestion // hydrolysishydrolysis AdjustAdjust pHpH toto approximatelyapproximately 1010 ExtractExtract withwith organicorganic solventsolvent 33 timestimes forfor highhigh recovery.recovery. ThisThis samplesample hashas highhigh backgroundbackground andand ionion-- suppression.suppression. ReconstituteReconstitute inin 2525 mMmM ammoniumammonium acetateacetate MIPMIP recoveriesrecoveries veryvery highhigh withwith lowlow backgroundbackground andand ionion--supression.supression. pH profile Zilpaterol 80 R 70 E 60 C 50 O V 40 E 30 R 20 Y 10 0 468101214 pH SolventSolvent ProfileProfile ofof ZilpaterolZilpaterol %% RecoveryRecovery pH Benzene Chloro- Methylene Ethyl Hexanes MTBE Pet Butanol Chloride Acetate Ether pH 22 9 48 87 0 61 1 11 PH 1 1 1 3 0 6 0 6 DetectionDetection GC/MSGC/MS (TMS(TMS derivative)derivative) –– ModerateModerate sensitivitysensitivity HPLCHPLC ColumnColumn TypeType –– CC--1818 columnscolumns givegive poorpoor resolution/retentionresolution/retention –– PhenylPhenyl--HexylHexyl excellentexcellent chromatographychromatography LC/MSLC/MS –– IonIon suppressionsuppression withwith urineurine andand tissues.tissues. Compound Mass Ion Fragmentor Prod Ion Collision Energy Rel Abundance Albuterol 239.15 240.2 95 148.1 16 100 77.1 60 25 222.1 4 15 51.1 104 9 Bitolterol 461.22 462.2 135 119.1 29 100 91.1 81 65 65.1 125 42 388.1 13 46 Clenbuterol 276.08 277.1 95 132.1 28 15 168 32 100 140 56 58 104.1 72 5 Fenoterol 303.15 304.2 110 107.1 33 100 77.1 77 37 51.1 117 14 135.1 17 83 Pirbuterol 240.15 241.2 110 167 13 100 185.1 9 51 94.1 49 65 149 21 83 Procaterol 290.16 291.2 110 273.2 8 100 162 28 32 77.1 88 2 174 36 4 Ractopamine 301.17 302.2 104 107 36 78 121 24 58 164.1 12 100 284.2 8 70 Terbutaline 225.14 226.1 94 107 42 99 77.1 42 68 125 42 96 152.1 42 100 Zilpaterol 261.15 262.2 105 185.1 24 100 157.1 36 30 244.1 8 78 130.1 52 32 ChromatographyChromatography C-18 3u 4x150 mm Phenyl-Hexyl 3u 4x150 mm CLENBUTEROL ZILPATEROL PROCATEROL ALBUTEROL ZilpaterolZilpaterol RetinaRetina PositivePositive Using combination of liq/liq and MIP extractions. High recovery with low background and low ion suppression SpecialSpecial ThanksThanks toto –– JimJim BrownBrown –– AndreAndre SalazarSalazar –– BrandyBrandy JessenJessen –– MarshaMarsha FarrarFarrar –– SylviaSylvia VegaVega –– ValsaValsa PhilipPhilip.