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Analytical Toxicology and The Horse By Thomas Tobin MVB, MRCVS, PhD, DABT University of Kentucky Maxwell H. Gluck Equine Research Center University of Kentucky SOUND HORSE CONFERENCE LOUISVILLE, KENTUCKY Friday, November 5th, 2010 ©Thomas Tobin, 2010 www.thomastobin.com (c) Thomas Tobin 2010 1 Equine Pharmacology, Therapeutics and Toxicology, Friday, November 5th, 2010, 9:00 AM www.thomastobin.com Thomas Tobin, Gluck Equine Research Center, [email protected] (c) Thomas Tobin 2010 2 (c) Thomas Tobin 2010 3 SUMMARY DRUG TESTING HIGHLY SENSITIVE 1.ELISA -> ENZYME LINKED IMMUNOSORBENT ASSAYS : FAST SCREENING 2. LC-MS-MS -> LIQUID CHROMATOGRAPHY MASS- SPECTROMETRY MASSSPECTROMETRY 3/ Detect PARTS PER TRILLION ©Thomas Tobin, 2010 4 IOWA DIVISION OF CRIMINAL INVESTIGATION CRIMINALISTICS LABORATORY DRUG IDENTIFICATION SECTION MICROCYSTALLINE TESTS Photo 10 Cocaine Photo 11 Methamphetamine (c) Thomas Tobin 2010 5 2/ ELISA TESTING • SIMPLE, FAST AND SENSITIVE • AN ELISA TEST IS BASICALY A HOME PREGNANCY TEST • RAPIDLY SCREEN FOR LOW PART PER BILLION CONCENTRATIONS ©(c) Thomas Thomas Tobin Tobin 2009 2010 6 (c) Thomas Tobin 2010 © Thomas Tobin 2009 7 (c) Thomas Tobin 2010 © Thomas Tobin 2009 8 Thomas Tobin 2010 ©(c) Thomas Tobin 2009 9 First introduction of ELISA Screening of Post-Race Urine, with Mass Spectral Analysis, Colorado, 1987 Sample Date # Urine Samples # Flagged by ELISA 10-3,4-87 34 5 3 Oxymorphone 10-4-87 16 1 1 Oxymorphone 10-11-87 8 1 1 Oxymorphone 10-17-87 36 3 2 Oxymorphone 10-17,18-87 27 3 1 Oxymorphone 10-20-87 21 4 4 Oxymorphone 10-27-87 24 1 1 Hydromorphone Totals, 9 Days 166 18 13 # Positive Drug Identified by GC/MS analysis (c) Thomas Tobin 2010 © Thomas Tobin 2009 10 NEOGEN ELISA KITS 2009 •Acetonide •Promazine •Alfentanil •Propoxyphene Group •Caffeine/Pentoxifylline Hydroxyzine 103610 •Ibuprofen •NEW •Ipratropium/Atropine •Isoxsuprine •Ketamine •Ketoprofen •Ketorolac •Levallorphan •Lidocaine •Lofentanil •Mazindol/Mazindol Metabolite •Meperidine •Mephentermine •Mepivacaine •Metaraminol •Methadone •Methamphetamine/MDMA •Methocarbamol •Methotrexate •Methylphenidate •Methylprednisolone •Nalbuphine •Nandrolone •Nikethamide •Opiate Group •Oxymorphone/Oxycodne •Pentazocine •Phenylbutazone •Procaine •Propranolol •Carfentanil Pyrilamine •Ractopamine •Reserpine •Sertraline •Stanozolol •Clenbuterol • •Cromoglycate •Sufentanil •Dantrolene •Sulfamethazine •Detomidine •THC •Dexamethasone •Theophylline •Dextromethorphan •Tramadol •NEW •Trenbolone •Diprenorphine TriamcinoloneAcetonide •Tricyclics Group •Zomepirac180210 •Doxapram •Clonidine/Romifi dine •NEW •Cocaine/Benzoylecgonine •Corticosteroid .Amphetamine • •Amphetamine •Butorphanol Ultra •Anileridine •Apomorphine/ Apocodeine •Azaperone •Barbiturate Group •Benzodiazepine Group NEW •Benzylpiperazine •Boldenone •Bronchodilator Group • Bumetanide •Buprenorphine Group •Droperidol •Ethacrynic Acid •Etorphine •Fenspiride •Fentanil Group •Fentanyl •Flunitrazepam •Flunixin •Fluoxetine •Fluphenazine •Furosemide •Glycopyrrolate •Guanabenz •Haloperidol •Haloperidol Metabolite NEW •Hydromorphone •Hydrochlorothiazide • (c) Thomas Tobin 2010 © Thomas Tobin 2009 11 FLORIDA DETECTIONS, 07/08 [18K] SUBSTANCE IDENTIFIED • • • • • • • • • • • • • • • • • • # of IDs ELISA ? Acepromazine metabolites ……………………12…... ELISA TEST Albuterol ………………………………………… 2 ….…ELISA TEST Benzoylecgonine………………..……………… 1 ….… ELISA TEST Butorphanol……………………………………… 3…..… ELISA TEST Caffeine/theophylline…………………………….8….…. ELISA TEST Cimetidine ………………..[MASKING]…………4…….. Clenbuterol……………………………………… 4…..… ELISA TEST DMSO……………………………………………… 5 Fentanyl……………………………………………3…..… ELISA TEST Glycopyrollate…………………………………… 5…… ELISA TEST Ipratropium…………………………………………2………ELISA TEST Lidocaine………………………………………… 1 …… ELISA TEST Methocarbamol……………………………………2 …… ELISA TEST Naproxen…………………………………………..1 Oxilofrine……[S. American drug]…………… 4 …… ELISA TEST Pentoxifylline…………………………………… 4 …… ELISA TEST Phenylbutazone…………………………………37……. ELISA TEST Ranitidine ……………………MASKING…….…4……… (c) Thomas Tobin 2010 © Thomas Tobin 2009 12 IMPACT OF ELISA TESTING SCREEN RAPIDLY AT HIGH SENSITIVITY FOR COMMONLY USED MEDICATIONS IN HORSES ©(c) Thomas Thomas Tobin Tobin 2009 2010 13 LIMITATIONS OF ELISA • An ELISA negative is, within the limits of the test, almost certainly a true negative • Any ELISA “positive” is just a “MAYBE” • Any ELISA data should be independently confirmed by what is usually Mass Spectral Confirmation Thomas Tobin 2010 ©(c) Thomas Tobin 2009 14 2/ Mass Spectral Analysis/Confirmation By Thomas Tobin MVB, MRCVS, PhD, DABT Maxwell H. Gluck Equine Research Center University of Kentucky SOUND HORSE CONFERENCE LOUISVILLE, KENTUCKY Friday, November 5th, 2010 ©(c) Thomas Thomas Tobin Tobin 2009 2010 15 LC-MS-MS LIQUID CHROMATOGRAPHY-MASS SPEC- MASS SPEC LC-MS-MS-> IDENTIFICATION of material. Further information: QUANTITY of material ©(c) Thomas Thomas Tobin Tobin 2009 2010 16 SCHEMATIC OF TANDEM MASS SPECTROMETRY (c) Thomas Tobin 2010 17 (c) © Thomas Thomas Tobin Tobin 2010 2009 18 METHOCARBAMOL MOLECULAR MASS 241.241 (c) © Thomas Thomas Tobin Tobin 2010 2009 19 LC-MS-MS 1/ FIRST MASS SPEC SELECTS 2/ PARENT ION ->241 -> Daughter ions? 3/ REACTION CHAMBER [slice and dice!] 4/ DAUGHTER IONS ->199 ->163 ->118 ©(c) Thomas Thomas Tobin Tobin 2009 2010 20 METHOCARBAMOL, LC-MS-MS (c) Thomas Tobin 2010 21 LC-MS-MS and Testing Sensitivity Liquid Chromatography-Mass Spectrometry- Mass Spectrometry: Microgram: parts per million: TLC Nanograms: parts per billion: ELISA Picograms: parts per trillion: LC-MS-MS Femtograms parts per quadrillion: LC-MS-MS TESTING IS NOW 1 MILLION FOLD MORE SENSITIVE THAN WHEN I STARTED IN THIS FIELD (c) Thomas Tobin 2010 22 3/ HOW LONG ARE DRUGS DETECTABLE FOR? ANSWER: DEPENDS 1/ DOSE 100,000 fold range 2/ HALF -LIFE 100-1,000 fold range 3/ SENSITIVITY 1,000,000 fold range OF TESTING ©(c) Thomas Thomas Tobin Tobin 2009 2010 23 DURATION OF MEDICATION ELIMINATION 1/ A dose of phenylbutazone contains 6X1021 molecules [stars in universe 6X1020 ] 2/ Half life ->7.22 hours, 3 half-lives will eliminate 50% plus 25% plus 12.5% = 90% / day. 3/ DRUG COMPLETLY ELIMINATED IN 21 DAYS ©(c) Thomas Thomas Tobin Tobin 2009 2010 24 (c) Thomas Tobin 2010 © Thomas Tobin 2009 25 DRUG ELIMINATION RULES • Number of drug molecules in a single administration is VERY large indeed. • Elimination continues long after the chemist fails to detect the medication. • RULE OF THUMB: Time to complete elimination is 60 half-lives, more or less. Thomas Tobin 2010 ©(c) Thomas Tobin 2009 26 SUMMARY DRUG TESTING HIGHLY SENSITIVE 1.ELISA -> ENZYME LINKED IMMUNOSORBENT ASSAYS : FAST SCREENING 2. LC-MS-MS -> LIQUID CHROMATOGRAPHY MASS- SPECTROMETRY MASSSPECTROMETRY 3/ Detect PARTS PER TRILLION ©Thomas Tobin, 2010 27 Acknowledgements The research presented here has been supported by grants from the Kentucky Horse Racing Commission and the Kentucky Equine Drug Research Council, The GraysonJockey Club Research Foundation, the National Institutes of Environmental Health Sciences, a gift from Mrs. John Hay Whitney and the following divisions of the Horsemen’s Benevolent and Protective Association: National, Canada, Florida, Nebraska, Kentucky, Ontario, Indiana, Charles Town, Ohio, Arkansas, Michigan, Pennsylvania, Alabama, Arizona, Iowa, Louisiana, Oklahoma, Tampa Bay Downs, Washington, Minnesota, Oregon, Texas and Mountaineer Park. The continuing support of the University of Kentucky, the Director and Faculty of the Gluck Equine Research Center and the University of Kentucky Equine Research Foundation are also gratefully acknowledged. ©(c) Thomas Thomas Tobin Tobin 2009 2010 28 SUMMARY 1/ DRUG/MEDICATION testing is now highly sensitive: it can detect PART PER TRILLION concentrations 3/ Allows highly sensitive testing for PERFORMANCE ALTERING SUBSTANCES: GO GET EM! 4/ THERAPEUTIC MEDICATIONS: limited sensitivity testing: DEFINED CONCENTRATION-“THRESHOLD” 4/ In the US we are developing THRESHOLDS for 55 THERAPEUTIC MEDICATIONS, Endogenous, Dietary and Environmental substances. 5/ THIS WILL STANDARDIZE TESTING IN THE US (c) Thomas Tobin 2010 © Thomas Tobin 2009 29 ©(c) Thomas Thomas Tobin Tobin 2009 2010 30 ©(c) Thomas Thomas Tobin Tobin 2009 2010 31 Example: Urinary Threshold For Lidocaine Lidocaine is excreted in horse urine as 3-OH LIDOCAINE GLUCURONIDE During analysis, the chemist hydrolyses the urine enzymatically and recovers 3-OH LIDOCAINE [regulatory analyte] We showed that urinary concentrations of 3-OH lidocaine < 300 ng/ml were unlikely to be associated with pharmacology, thereby establishing the scientific basis for a regulatory threshold. We have synthesized and made available to racing chemists the required 3-OH lidocaine reference standard and the corresponding deuterated internal standard, as summarized on the next slide. © Thomas (c) Thomas TobinTobin January, 20102005 32 © Thomas (c) Thomas TobinTobin January, 20102005 33 Thomas Tobin 2010 ©(c) Thomas Tobin 2009 34 SUMMARY • DRUG TESTING IS NOW HIGHLY SENSITIVE • [ELISA]: ENZYME LINKED IMMUNOSORBENT ASSAYS FAAAAAAST ------ SCREENING • [LC-MS-MS] LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY MASS SPECTROMETRY • CAN UNEQUIVOCALLY IDENTIFY AND QUANTIFY PICOGRAMS/ML, OR LOW PART PER TRILLION CONCENTRATIONS IN BLOOD OR URINE OR IN TOPICAL SWABS : HIGHLY SPECIFIC AND RELIABLE • SHOULD BE POSSIBLE TO TAKE TOPICAL SWABS OR BLOOD SAMPLES AND DETECT RECENT APPLICATION OF MOST SMALL MOLECULE AGENTS (c) Thomas Tobin 2010 35 LOCAL ANESTHETIC “NO EFFECT” THRESHOLDS • • • • • Procaine……………….………..50 ppb in urine 3-Hydroxylidocaine…………….310 ppb in urine 3-hydroxymepivacaine…………65 ppb in urine 3-hydroxybupivacaine………….28 ppb in urine Benzoylecgonine……………….165 ppb in urine Note that what we detect in urine is often a unique urinary metabolite, for which we have synthesized the required reference standards. ©(c) Thomas Thomas Tobin Tobin 2009 2010 36