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


mailto:[email protected]

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


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

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



10-17,18-87 27 3 1 Oxymorphone


10-27-87 24 1 1 Hydromorphone

Totals, 9 Days 166 18 13

© Thomas Tobin 2009


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 •


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………


IMPACT OF ELISA TESTING

SCREEN

RAPIDLY AT HIGH SENSITIVITY FOR COMMONLY USED

MEDICATIONS IN HORSES


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


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


LC-MS-MS

LIQUID CHROMATOGRAPHY-MASS

SPEC- MASS SPEC

LC-MS-MS-> IDENTIFICATION of material.

Further information: QUANTITY of material



SCHEMATIC OFTANDEM MASS SPECTROMETRY

http://en.wikipedia.org/wiki/File:MS_MS.png


© Thomas Tobin 2009

METHOCARBAMOLMOLECULAR MASS

241.241


http://en.wikipedia.org/wiki/File:Methocarbamol_structure.svg

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



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





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



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


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.


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.


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 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.


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


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.


analytical toxicology and the horse by thomas tobin mvb, mrcvs, phd, dabt university of kentucky...

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