Analytical Toxicology and The Horse By Thomas Tobin MVB, MRCVS, PhD, DABT University of Kentucky...
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Transcript of Analytical Toxicology and The Horse By Thomas Tobin MVB, MRCVS, PhD, DABT University of Kentucky...
Analytical Toxicology and The Horse
By
Thomas TobinMVB, MRCVS, PhD, DABT
University of KentuckyMaxwell H. Gluck Equine Research Center
University of Kentucky SOUND HORSE CONFERENCE
LOUISVILLE, KENTUCKY
Friday, November 5th, 2010©Thomas Tobin, 2010
www.thomastobin.com
1(c) Thomas Tobin 2010
Thomas Tobin, Gluck Equine Research Center, [email protected]
Equine Pharmacology, Therapeutics and
Toxicology, Friday, November 5th, 2010, 9:00 AM
www.thomastobin.com
2(c) Thomas Tobin 2010
3(c) Thomas Tobin 2010
SUMMARYDRUG TESTING HIGHLY SENSITIVE
1.ELISA -> ENZYME LINKED IMMUNOSORBENT
ASSAYS : FAST SCREENING
2. LC-MS-MS -> LIQUID CHROMATOGRAPHY
MASS- SPECTROMETRY MASS- SPECTROMETRY
3/ Detect PARTS PER TRILLION
4©Thomas Tobin, 2010
Photo 10 Cocaine
Photo 11 Methamphetamine
IOWA DIVISION OF CRIMINAL INVESTIGATIONCRIMINALISTICS LABORATORY
DRUG IDENTIFICATION SECTION
MICROCYSTALLINE TESTS
5(c) Thomas Tobin 2010
2/ ELISA TESTING
• SIMPLE, FAST AND SENSITIVE
• AN ELISA TEST IS BASICALY A HOME PREGNANCY TEST
• RAPIDLY SCREEN FOR LOW PART PER BILLION CONCENTRATIONS
© Thomas Tobin 2009 6(c) Thomas Tobin 2010
© Thomas Tobin 20097(c) Thomas Tobin 2010
© Thomas Tobin 20098(c) Thomas Tobin 2010
© Thomas Tobin 2009 9(c) Thomas Tobin 2010
First introduction of ELISA Screening of Post-Race Urine, with Mass Spectral
Analysis, Colorado, 1987 Sample Date # Urine
Samples# Flagged by ELISA
# Positive by GC/MS analysis
Drug Identified
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
© Thomas Tobin 2009
10(c) Thomas Tobin 2010
NEOGEN ELISA KITS 2009
•Acetonide •Alfentanil 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
•Promazine Group •Propoxyphene •Propranolol Pyrilamine •Ractopamine •Reserpine •Sertraline •Stanozolol •16Hydroxystanozolol •Sufentanil •Sulfamethazine •THC •Theophylline •Tramadol •Trenbolone TriamcinoloneAcetonide •Tricyclics Group •Zomepirac180210
•.Amphetamine •Amphetamine Ultra •Anileridine •Apomorphine/ Apocodeine •Azaperone •Barbiturate Group •Benzodiazepine Group NEW •Benzylpiperazine •Boldenone •Bronchodilator Group • Bumetanide •Buprenorphine •
•Butorphanol •Caffeine/Pentoxifylline •Carfentanil •Clenbuterol •Clonidine/Romifi dine •NEW •Cocaine/Benzoylecgonine •Corticosteroid Group •Cromoglycate •Dantrolene •Detomidine •Dexamethasone •Dextromethorphan •NEW •Diprenorphine •Doxapram •Droperidol •Ethacrynic Acid •Etorphine •Fenspiride •Fentanil Group •Fentanyl •Flunitrazepam •Flunixin •Fluoxetine •Fluphenazine •Furosemide •Glycopyrrolate •Guanabenz •Haloperidol •Haloperidol Metabolite •Hydrochlorothiazide NEW •Hydromorphone •
© Thomas Tobin 2009 11(c) Thomas Tobin 2010
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………
© Thomas Tobin 200912(c) Thomas Tobin 2010
IMPACT OF ELISA TESTING
SCREEN
RAPIDLY AT HIGH SENSITIVITY FOR COMMONLY USED
MEDICATIONS IN HORSES
© Thomas Tobin 2009 13(c) Thomas Tobin 2010
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 2009 14(c) Thomas Tobin 2010
2/ Mass Spectral Analysis/Confirmation
ByThomas Tobin
MVB, MRCVS, PhD, DABTMaxwell H. Gluck Equine Research Center
University of Kentucky
SOUND HORSE CONFERENCELOUISVILLE, KENTUCKY
Friday, November 5th, 2010
© Thomas Tobin 2009 15(c) Thomas Tobin 2010
LC-MS-MS
LIQUID CHROMATOGRAPHY-MASS
SPEC- MASS SPEC
LC-MS-MS-> IDENTIFICATION of material.
Further information: QUANTITY of material
© Thomas Tobin 2009 16(c) Thomas Tobin 2010
17(c) Thomas Tobin 2010
SCHEMATIC OFTANDEM MASS SPECTROMETRY
© Thomas Tobin 2009 18(c) Thomas Tobin 2010
© Thomas Tobin 2009
METHOCARBAMOLMOLECULAR MASS
241.241
19(c) Thomas Tobin 2010
LC-MS-MS1/ FIRST MASS SPEC SELECTS
2/ PARENT ION ->241 -> Daughter ions?
3/ REACTION CHAMBER [slice and dice!]
4/ DAUGHTER IONS ->199 ->163 ->118
© Thomas Tobin 2009 20(c) Thomas Tobin 2010
21(c) Thomas Tobin 2010
METHOCARBAMOL, LC-MS-MSH
H
H
H
O
O
OH
O
H2N O
H3C
H
H
H
H
O
O
OH
HOH3C
Chemical Formula: C10H14O4•+
Exact Mass: 198.09
H
H
H
H
O
O
OH
O
H2N O
H3C
H
H
H
H
O
O
OH
O
H2N O
H3C
Chemical Formula: C4H8NO3•
Exact Mass: 118.05
H
H
H
H
O
O
OH
O
H2N O
H3C
H
H
H
H
O
O
OH
O
H2N O
H3C
H
H
H
H
O
O
OH
O
H2N O
H3C
Chemical Formula: C11H14NO4•
Exact Mass: 224.09Chemical Formula: C10H12O2
2•
Exact Mass: 164.08
2+
Chemical Formula: C11H15NO5
Molecular Weight: 241.24
Chemical Formula: C11H15NO5
Molecular Weight: 241.24
Chemical Formula: C11H15NO5
Molecular Weight: 241.24
D
D
D
D
O
O
OH
O
H2N O
H3C
D
D
D
D
O
O
OH
HOH3C
Chemical Formula: C10H10D4O4
Molecular Weight: 202.24Chemical Formula: C11H11D4NO5
Molecular Weight: 245.27
LC-MS-MS and Testing Sensitivity
Liquid Chromatography-Mass Spectrometry- Mass Spectrometry:
Microgram: parts per million: TLC Nanograms: parts per billion: ELISAPicograms: 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 22(c) Thomas Tobin 2010
23
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© Thomas Tobin 2009 23(c) Thomas Tobin 2010
24
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
© Thomas Tobin 2009 24(c) Thomas Tobin 2010
25© Thomas Tobin 2009
25(c) Thomas Tobin 2010
26
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 2009 26(c) Thomas Tobin 2010
SUMMARYDRUG TESTING HIGHLY SENSITIVE
1.ELISA -> ENZYME LINKED IMMUNOSORBENT
ASSAYS : FAST SCREENING
2. LC-MS-MS -> LIQUID CHROMATOGRAPHY
MASS- SPECTROMETRY MASS- SPECTROMETRY
3/ Detect PARTS PER TRILLION
27©Thomas Tobin, 2010
28
Acknowledgements
The research presented here has been supported by grants from the Kentucky Horse Racing Commission and the Kentucky Equine Drug Research Council, The Grayson-Jockey 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.
© Thomas Tobin 2009 28(c) Thomas Tobin 2010
SUMMARY1/ 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
© Thomas Tobin 200929(c) Thomas Tobin 2010
30© Thomas Tobin 2009 30(c) Thomas Tobin 2010
31© Thomas Tobin 2009 31(c) Thomas Tobin 2010
© Thomas Tobin January, 2005
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.
32(c) Thomas Tobin 2010
© Thomas Tobin January, 2005 33(c) Thomas Tobin 2010
34© Thomas Tobin 2009 34(c) Thomas Tobin 2010
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
35(c) Thomas Tobin 2010
36
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.
© Thomas Tobin 2009 36(c) Thomas Tobin 2010