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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
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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
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(c) Thomas Tobin 2010
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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
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IOWA DIVISION OF CRIMINAL INVESTIGATION
CRIMINALISTICS LABORATORY
DRUG IDENTIFICATION SECTION
MICROCYSTALLINE TESTS
Photo 10
Cocaine
Photo 11
Methamphetamine
(c) Thomas Tobin 2010
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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
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Tobin
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(c) Thomas Tobin 2010
© Thomas Tobin 2009
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(c) Thomas Tobin 2010
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Thomas
Tobin
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©(c)
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Tobin
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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
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5
3
Oxymorphone
10-4-87
16
1
1
Oxymorphone
10-11-87
8
1
1
Oxymorphone
10-17-87
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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
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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
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© Thomas
Tobin
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FLORIDA DETECTIONS, 07/08 [18K]
SUBSTANCE IDENTIFIED
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# 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
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IMPACT OF ELISA TESTING
SCREEN
RAPIDLY AT HIGH SENSITIVITY
FOR COMMONLY USED
MEDICATIONS IN HORSES
©(c)
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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
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Tobin
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©(c)
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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
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2010
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LC-MS-MS
LIQUID CHROMATOGRAPHY-MASS
SPEC- MASS SPEC
LC-MS-MS-> IDENTIFICATION
of
material.
Further information: QUANTITY of
material
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Thomas
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SCHEMATIC OF
TANDEM MASS SPECTROMETRY
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(c)
© Thomas
Thomas Tobin
Tobin 2010
2009
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METHOCARBAMOL
MOLECULAR MASS
241.241
(c)
© Thomas
Thomas Tobin
Tobin 2010
2009
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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)
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METHOCARBAMOL, LC-MS-MS
(c) Thomas Tobin 2010
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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
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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
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Tobin
Tobin
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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)
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(c) Thomas Tobin 2010
© Thomas Tobin 2009
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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
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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
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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
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Tobin
Tobin
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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
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©(c)
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©(c)
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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
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© Thomas
(c) Thomas
TobinTobin
January,
20102005
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Thomas
Tobin
2010
©(c)
Thomas
Tobin
2009
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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
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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
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Tobin
Tobin
2009
2010
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