Detection of Various Performance Enhancing Substances in Specimens Collected from Race Horses in Illinois: A Five-Year Experience *

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1 Detection of Various Performance Enhancing Substances in Specimens Collected from Race Horses in Illinois: A Five-Year Experience * Lisa Taddei, Marc Benoit, Andre Sukta, Joshua Peterson, R.E. Gaensslen, and Adam Negrusz Animal Forensic Toxicology Laboratory, University of Illinois at Chicago, 2242 West Harrison Street, Chicago, Illinois Abstract In order to protect the integrity of horse racing in Illinois, a complex testing of urine and blood specimens collected post-race from winning and special designation horses is continuously conducted. The initial screening by immunoassays was followed by the confirmation on presumptive positive samples. Instrumental screening was also conducted. Perimortem and postmortem specimens and special exhibits (syringes, needles, etc.) were also analyzed. The administration of alkalinizing agents was detected by measuring the total plasma carbon dioxide concentration. The laboratory analyzed specimens collected post race from winning horses and special designation horses at eight race tracks in the State of Illinois over the five-year time period ( ). The total number of specimens collected was 91,808, comprising 45,210 urine specimens and 46,598 blood specimens. The total number of violations was 413 (0.45% of the total number of specimens analyzed); 207 were blood specimens (0.44% of the total blood specimens analyzed), and 206 were urine specimens (0.45% of the total urine specimens analyzed). A total of 220 violations were reported for harness horses, and 193 were reported for Thoroughbred horses. The number of reported violations of the total tested specimens in Illinois was small, but a wide variety of performance-enhancing drugs was shown. Introduction * This work was presented at the 2011 AAFS meeting in Chicago, Illinois. Author to whom correspondence should be addressed. anegrusz@uic.edu. One of the most important objectives of each horseracing jurisdiction is to protect the integrity of the sport through the administration of a multifaceted control of the use of substances that have a potential to enhance a horse s performance in a race. The drugs and substances that have been identified as performance enhancing have been completely and comprehensively classified based upon pharmacological activity. Within these guidelines the Association of Racing Commissioners International (ARCI) has established five classes of performance enhancing substances ranging from Class 1 Substances, those demonstrating the greatest potential to enhance performance, to Class 5 Substances, those demonstrating the least potential for enhancing performance (1). The ARCI definitions for each class follows. Class 1: Stimulant and depressant drugs that have the highest potential to affect performance and that have no generally accepted medical use in the racing horse. Many of these agents are Drug Enforcement Administration Schedule II substances. Examples include opioids, cocaine, and amphetamines. Class 2: Includes drugs that have a high potential to affect performance, but less than those drugs in Class 1. Drugs in Class 2 are 1. not generally accepted as therapeutic agents in racing horses or 2. therapeutic agents that have a high potential for abuse. Class 3: Drugs in that group may or may not have generally accepted medical use in the racing horse, but the pharmacology of which suggests less potential to affect performance than drugs in Class 2. Examples include anabolic steroids, bronchodilators, local anesthetic, or high-ceiling (loop) diuretics. Class 4: This class includes therapeutic medications that have less potential to affect performance than those in Class 3. Examples of drugs in this class include less potent diuretics acting at distal convoluted tubule (e.g., chlorothiazide), or acting at collecting duct system (e.g., spironolactone), corticosteroids, antihistamines, or skeletal muscle relaxants without prominent central nervous system effects, non-steroidal antiinflammatory drugs (NSAIDs), and others. Class 5: This class includes those therapeutic medications for which concentration limits have been established by the racing jurisdictions, as well as certain miscellaneous agents such as dimethyl sulfoxide (DMSO) and other medications as deter- 438 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher s permission.

2 Table I. Threshold Concentrations for Selected Compounds in Specimens Collected from Race Horses in Illinois Substance Specimen Threshold Phenylbutazone and/or metabolite Serum 5 µg/ml (oxyphenylbutazone) Furosemide Serum 100 ng/ml Total Carbon dioxide Plasma 37 mmol/l Flunixin Serum 20 ng/ml Ketoprofen Serum 10 ng/ml Benzoylecgonine (cocaine metabolite) Urine 150 ng/ml* DMSO Urine 500 µg/ml Isoxsuprine Urine 1000 ng/ml Pyrilamine Urine 50 ng/ml * Every concentration equal to or greater than the LOQ is reported as a violation. Levels equal to or greater than the threshold are associated with more severe penalties. mined by the regulatory bodies. Included specifically are agents that have very localized actions only, such as anti-ulcer drugs, and certain anti-allergic drugs. The anticoagulant drugs are also included. The ARCI guidelines were developed to aid in the interpretation of racehorse drug-screening results and include suggested penalties for each class violation. These guidelines were also designed to give racing authorities, such as racing regulators or stewards, a framework within which they can evaluate the relative performance enhancing effects of various drugs and substances and the associated impact that a drug or substance may be anticipated to have on the outcome of a race (2). The penalty guidelines allow for mitigating circumstances to be taken into account when the racing authorities issue a ruling dictated by the Illinois Administrative Code (3). A penalty in Illinois may include anything from a small fine, to the monetary prize (purse money) redistribution, to, in the most severe cases, a racing ban. All Illinois Racing Board rules and regulations regarding the administration of medication and testing for the presence of performance enhancing drugs are published in the General Assembly's Illinois Administrative Code, Part 603 Medication (3). The Rule lists threshold concentrations for medications permitted for therapeutic administration in blood (serum, plasma) such as phenylbutazone, furosemide, flunixin, ketoprofen, selected anabolic steroids (testosterone, boldenone, nandrolone, stanozolol), thresholds for selected medications in urine (e.g., isoxsuprine, DMSO, anabolic steroids), as well as O- desmethylpyrilamine (pyrilamine metabolite) and benzoylecgonine (cocaine metabolite). The Rule also requires testing for the use of alkalinizing agents, such as sodium bicarbonate, the employment of which is referred to as milkshaking. The use of alkalinizing agents is revealed by measuring the total carbon dioxide (TCO 2 ) concentration in plasma (4 6). The list of all thresholds is presented in Table I. There is a zero tolerance established for Class 1 3 drugs. If Class 4 or 5 drugs are detected in a specimen, quantitation of all relevant drugs must be performed and the subsequent results reported to the Illinois Racing Board Legal Council, state veterinarians, and stewards. The only comprehensive literature report on drug use in racing dates from the 12-year period between 1970 through 1981 (7). This paper describes the principles of equine testing for performance-enhancing substances, including allowed medications, and presents a summary of analytical findings on all winning racehorses obtained during the five-year period (2004 through 2009) in Illinois, the most frequently encountered drug violations, and drug use trends in general. Methods Specimen collection from race horses The Animal Forensic Toxicology Laboratory analyzed a total of 91,808 specimens, 45,210 urine specimens and 46,598 blood specimens, collected from Thoroughbred and harness horses at eight race tracks in Illinois over the five-year time period from July 2004 through June The results from only seven race tracks are presented in this report because no violations were recorded on one track. Blood samples were drawn only by the State Veterinarian after checking the horses identification tattoos. Blood from horses was collected to 13-mL serum separator and 4.5 plasma-separator Vacutainer tubes. Plasma samples were collected for the TCO 2 and erythropoietin (EPO) analysis. According to the current testing protocol in Illinois, only harness horses are tested for the use of alkalinizing agents, with the exception of graded stakes races. The main argument for testing harness horses and not Thoroughbred for TCO 2 is the belief that this type of blood doping will only provide a benefit to horses competing in endurance races. A graded stakes race term was adopted nationwide by the Thoroughbred Owners and Breeders Association (TOBA) to Thoroughbred horse races where the stake, or entry fee, must be paid by owners. The race track then adds an additional amount which constitutes the total amount of prize money to be paid to the first, second, third, and typically fourth place finishers. These races provide approximately 100 blood and urine samples for analysis in Illinois annually. No elevated TCO 2 plasma concentrations were encountered during the time period. The horse s name (later covered by a seal to hide information from the laboratory personnel), race number, and winner/special designation were written on the permanent label of the blood tubes. Special designation horses are usually the second or third place finishers selected by stewards or state veterinarians for random drug testing. Some equine athletes may also be flagged for drug testing because of unusual behavior or surrounding circumstances. After specimen collection, the blood tubes were placed upright in a tube rack for between 15 to 30 min to allow for blood clotting to progress and then centrifuged for 15 min to allow phase separation to occur. Following the separation of 439

3 serum or plasma and red blood cells in the centrifuge, each blood tube was sealed by the Chief Veterinary Technician. One serum and one plasma tube from each set was marked as a referee specimen; the remaining samples were marked as laboratory samples. Once sealed, the tubes were placed in the locked refrigerator and held there until placed in the sealed sample box for shipment to the laboratory. Urine samples were collected by the State Veterinary Technicians using a long-handled plastic cup lined with a disposable plastic collection bag. The natural void urine from the horse was collected in the secured stall area. Following sample collection, the urine sample was transferred from the disposable plastic liner bag into two plastic urine containers in equal portions and sealed. One container was marked as laboratory, and the other as a referee sample. According to the Illinois Rule, when an unauthorized substance or an overage of an authorized medication is detected in a laboratory sample, the horse owner, trainer, or other person charged with a violation has an opportunity to request that the referee sample be opened and a split portion sent to another laboratory to verify the positive test. The remaining portion of a referee sample shall be preserved by the laboratory in case further testing is requested (3). When all blood and urine samples had been collected for the day s racing program, they were placed in a shipping container along with the proper paperwork, sealed, and shipped to the laboratory according to the chain-of-custody procedures. Perimortem and postmortem samples The Animal Forensic Toxicology Laboratory analyzed a total of 302 blood samples collected from animals awaiting euthanization (perimortem) or postmortem over the five-year time period (2004 through 2009). Special exhibits Over the five-year period of time ( ) the Animal Forensic Toxicology Laboratory analyzed 116 specimens identified as special exhibits. These were items such as syringes, needles, neat drugs, empty containers and vials, liquids, plastic bags found on race courses and adjacent properties and/or confiscated from persons within the surrounding confines of racecourses by racing authorities and subsequently delivered under chain of custody to the laboratory for analysis. Initial screening All urine and blood samples collected post-race from winning horses in Illinois were initially screened for the presence of performance enhancing substances by using at least 65 commercially available enzyme-linked immunosorbent assay (ELISA) kits. The laboratory uses group-specific ELISA kits designed to detect several drugs within one pharmacological group (e.g., bronchodilators, antihistamines, opioids) and a single analyte (e.g., indometacin-specific kit). The laboratory also screened the specimens collected perimortem, postmortem, and the special exhibits for the presence of prohibited substances. The initial screening in equine testing cannot always be performed by employing immunochemical methodologies including ELISA. The main reasons include insufficient sensitivity of available kits, a high percentage of 440 false-positive results, a very high cost per test, or there is no commercially available kit. For the above reasons the instrumental screening was also performed employing the established solid-phase or solvent extraction procedures followed by gas chromatography mass spectrometry (GC MS) (e.g., DMSO analysis), or ion trap or triple-quadrupole liquid chromatography (LC) MS (e.g., anabolic steroids analysis) methods. All samples selected by instrumental screening as presumptive positive were reanalyzed by either the same method with the different scan mode parameters, or by different instrumental technique. For anabolic steroids, for example, the initial LC MS MS analysis was performed using one ion transition. In the second analysis, at least five transitions were considered for drug identification. Positive drug identification was always based on analysis of at least two portions of the original sample. In addition, the identification of a substance always comprised a direct comparison with a reference standard added to a corresponding naïve biological specimen and analyzed in series with the test sample. Confirmation All presumptive positive samples selected by ELISA screening were subsequently confirmed by GC MS or LC MS( MS) methods according to the standard operating procedures (SOPs). All quantitative analyses were also performed according to the respective SOPs. Table II. Total Number of Specimens Tested and Percentage of Reported Violations in Blood and Urine ( ) Specimen Number of Samples Percentage of Total Blood 46, Urine 45, Total 91, Violations in blood Violations in urine Total violations (blood, urine) Table III. Number of Perimortem and Postmortem Samples and Special Exhibits Tested ( ) Year Perimortem/Postmortem Special Exhibits Total

4 The use of alkalinizing agents, such as sodium bicarbonate, was revealed primarily by means of the measurement of the plasma TCO 2 concentration as well as the additional information provided by measuring plasma total proteins and the ion concentrations for K +, Na +, Cl, and Ca +2. The analysis was performed according to the existing SOP by using Beckman SYNCHRON EL-ISE instrument, and since January 1, 2009, the laboratory has been performing these analyses using the Beckman Coulter DxC 600 analyzer. If the initial TCO 2 measurement in the laboratory portion of plasma indicated an elevated concentration (equal to or higher than 37 mmol/l), it was the Illinois Racing Board recommendation that the referee portion of plasma sample be opened and analyzed by the laboratory. Results and Discussion During the 5-year period of time ( ), the laboratory analyzed a total of 91,808 specimens (45,210 urine samples and 46,598 blood samples) collected post-race from winning and special designation (horses other than those which took first place) Thoroughbred and harness horses, 302 perimortem and postmortem blood samples, and 116 special exhibits from 7 race tracks in the State of Illinois. The total number of specimens tested and the percentage of reported violations (confirmed presence of an unauthorized performance enhancing substance, or an overage of an authorized medication) in blood and in urine are presented in Table II. Table III presents the total number of special exhibits and perimortem and postmortem blood samples tested. The total number of violations reported was 413 (0.45% of the total number of specimens analyzed) including 207 drug-positive blood specimens (0.44% of all blood samples analyzed) and 206 urine samples (0.45% of all urine specimens analyzed). Two-hundred twenty violations were reported for harness horses and 193 for Thoroughbred horses. To date, there has only been a single report in the scientific literature known to the authors concerning the relative proportion of drug-positive samples to the total number of samples collected during the 12-year period between 1970 through 1981 in Great Britain including comparisons with Table IV. Number of Positive Reports by Year and Race Track ( ) Race Track other countries over varying periods between 1975 and 1981 (7). The proportion of positive results found in that study was one in 400 (0.25%) for both winning and non-winning horses. Drugs such as methylxanthines, NSAIDs, local anesthetics, and anabolic steroids were the most frequently detected. The author concluded that based on the evidence presented, deliberate doping in Great Britain was relatively rare (7). In the study presented here the number of positive reports issued to the authorities by year and race track in Illinois is presented in Table IV. The number of reported violations ranged from 123 (2006) to 40 two years later (2008). The increased intensity of testing and reporting most likely led to decreased doping and therefore a decreased number and percentage of positive results, and as a consequence, a decreased number of reports to the State Authorities. The most frequently identified drugs are listed in Table V. In addition to the findings detailed in Table V, one violation was reported for each of the following drugs: acetaminofen, buprenorphine, carprofen, chlorpromazine, codeine, desipramine, fluoxetine, glycopyrolate, guaifenesin, hydromorphone, imipramine, meperidine, mepivacaine, methamphetamine, nalbuphine, nalorphine, oxazepam, oxymorphone, phenobarbital, phentermine, prednisolone, prednisone, promazine, tramadol, and verapamil. As Table V clearly shows, the most frequent violations include the overages (concentrations of permitted medications equal to or exceeding the set threshold) of NSAIDs such as phenylbutazone with its metabolite oxyphenylbutazone (a total of 111) and flunixin (44 cases). Because both drugs are allowed Class 4 medications, there are thresholds established for phenylbutazone and flunixin (Table I), and therefore their concentrations in blood have to be monitored to differentiate acceptable therapeutic use versus administration for performance enhancing effects (8,9). The laboratory also found several other non-steroidal anti-inflammatory and analgesic drugs including diclofenac, indometacin, naproxen, ketorolac, acetaminophen, and carprofen, but with much lower frequency than phenylbutazone and flunixin. Over the five-year period of time ( ), the number of reported violations with furosemide was high on the list (total of 25 violations) (Table V). Furosemide is a rapidly acting loop diuretic approved by the U.S. racing industry for treatment of exercise-induced pulmonary hemorrhage (EIPH) Year Arlington Fairmount Hawthorne Balmoral Maywood DuQuoin Springfield Total Total

5 (10,11). The use of furosemide is controversial and broadly criticized because of its potential to enhance the performance of race horses (11). During the same five-year period of time, the laboratory detected and reported 33 TCO 2 violations with the total carbon dioxide post-race plasma concentrations ranging from 37 to 42 mmol/l. Typically the alkalinizing agent (e.g., sodium bicarbonate) is administered to the horse by gavage to hypothetically titrate lactic acid produced in muscles during the excessive exercising, resulting in elevated TCO 2 concentration in blood. In one study, it was determined that the stabled horses had a mean TCO 2 level of 30 mmol/l (standard deviation 1.2) and the concentrations were normally distributed, whereas the pre-race TCO 2 concentrations were not. The authors concluded that it is extremely unlikely for a normal horse to have a resting TCO 2 concentration above 36 mmol/l (4). There are few factors, such as the use of furosemide, sex, class and distance of race, and weather, that may be associated with the prerace TCO 2 concentration in race horses (5,6). A broad review of milkshaking practices and its physiological and analytical consequences was published by Kline (12). In TCO 2 testing scenario in Illinois, plasma samples are collected 90 min postrace when all physiological post-exercise parameters come back to baseline. If the TCO 2 concentration is elevated, a horse is typically quarantined and TCO 2 concentration monitored to rule out naturally occurring high TCO 2 plasma levels. Cocaine is classified as Class 1 drug according to the ARCI Uniform Classification Guidelines for Foreign Substances (1), and has a very high potential for impacting the outcome of a race. During the 5-year period of time ( ), the laboratory detected benzoylecgonine (the primary inactive metabolite of cocaine) in 34 urine specimens. In 2005, the Illinois Racing Board issued new medication rules establishing the threshold level of 150 ng/ml for benzoylecgonine in equine urine (3). The ruling was associated with the possibility of environmental contamination with cocaine and the potential for exposure of race horses to trace levels of the drug, producing low levels of benzoylecgonine in horse urine. The issues of low benzoylecgonine levels (below 150 ng/ml) in equine urine, their sources, and the consequences and potential impact on a race, were addressed by this group elsewhere (13). None of the benzoylecgonine levels reported in this study exceeded 100 ng/ml. The other most frequent violations include DMSO and ergonovine (Table V). DMSO is an organic solvent used in horses as a liniment and for the treatment of increased intracranial pressure and/or cerebral edema. It can also be applied to the skin to increase the cooling of body temperature. The threshold for DMSO in urine established by the Illinois Racing Board is 500 ug/ml (Table I). Ergonovine (ergometrine) is a potent ergot derivative similar to LSD acting on alpha-adrenergic, dopamine, and serotonin (5-HT 2 ) receptors. The laboratory found a total of 21 cases positive for this drug after obtaining information from harness track personnel of possible abuse. The laboratory was able to move quickly and add ergonovine to the ELISA screening and develop a semi-quantitative GC MS confirmatory procedure. The other groups of drugs detected in Illinois include antihistamines (pyrilamine, anti-asthmatic cromolyn), vasodilators (isoxsuprine, verapamil), muscle relaxants (methocarbamol), tranquilizers (acepromazine, promazine, chlorpromazine), local anesthetics (procaine, lidocaine, mepivacaine), anticholinergic (glycopyrolate), and opioid analgesics (morphine, tramadol, hydromorphone, nalbuphine, codeine, buprenorphine, nalorphine, oxymorphone). In addition, the laboratory previously reported two cases of etorphine (an extremely potent morphine analogue) administration to race horses (14). In a few cases, antidepressants (fluoxetine, imipramine, desipramine), benzodiazepines (oxazepam), glucocorticosteroids (prednisone, prednisolone), and even methamphetamine and phenobarbital were detected. Table V. Drugs and Substances Most Frequently Reported ( ) Drug Thoroughbred Harness Total Phenylbutazone and oxyphenylbutazone* Flunixin Cocaine (benzoylecgonine) Total Carbon dioxide Furosemide Ergonovine DMSO Pyrilamine (O-desmethylpyrilamine) Cromolyn Diclofenac Indometacin Isoxsuprine Acepromazine [2-(1-hydroxyethyl) promazine]" Methocarbamol Procaine Naproxen Ketorolac Etorphine Lidocaine (3-hydroxylidocaine) # Morphine * Pharmacologically active metabolite of phenylbutazone. Cocaine metabolite. Thoroughbred horses are not tested for TCO 2. Pyrilamine metabolite. " Acepromazine metabolite. # Lidocaine metabolite. 442

6 Conclusions The number of reported violations of the total tested specimens in the State of Illinois was small, but a wide variety of performance-enhancing drugs and substances was shown. The most frequently reported violations include permitted therapeutic medications (phenylbutazone, flunixin). The authors conclude that those drugs having a valid medical indication and are commonly administered to horses also exhibit the greatest potential for being abused. Low levels of benzoylecgonine in horse urine most likely reflect a casual contact of a horse with cocaine-contaminated objects. The authors conclude that, based on the pharmacokinetics of cocaine in horses and a minimum effective cocaine dose capable of improving horse s performance, the low levels detected in this study are not indicative of a deliberate doping. For many medications it would be difficult to conclude whether a drug was administered to a horse for the purpose of doping or the amount detected was residual. Finally, it is the authors conclusion that the most important steps in developing and maintaining a successful equine forensic toxicology program for the detection of doping with performance enhancing drugs are: communication with race track personnel, staying current on regional and global trends in doping and research and the ability to adjust one s program to meet current needs. Acknowledgment This work was funded by the Contract Number CC from the Illinois Racing Board, Illinois Department of Revenue. References 1. Association of Racing Commissioners International, Inc., Drug Testing Standards and Practices Program Model Rules Guidelines, Uniform Classification Guidelines for Foreign Substances and Recommended Penalties and Model Rule, com/druglisting.pdf (accessed January 2011). 2. C.R. Short, R.A. Sams, L.R. Soma, and T. Tobin. The regulation of drugs and medicines in horse racing in the United States. The Association of Racing Commissioners International Uniform Classification of Foreign Substances Guidelines. J. Vet. Pharmacol. Ther. 21(2): (1998). 3. The General Assembly s Illinois Administrative Code, Title 11: Alcohol, Horse Racing, and Lottery Subtitle b: Horse Racing Chapter I: Illinois Racing Board Subchapter C: Rules Applicable to all Occupation Licensees, Part 603 Medication, sections.html (2011). 4. D.E. Auer, K.V. Skelton, S. Tay, and F.C. Baldock. Detection of bicarbonate administration (milkshake) in standardbred horses. Aust. Vet. J. 70(9): (1993). 5. N.D. Cohen, S.D. Stanley, R.M. Arthur, and N. Wang. Factors influencing pre-race serum concentrations of total carbon dioxide in Thoroughbred horses racing in California. Equine Vet. J. 38(6): (2006). 6. K. Kline, D. Fitzpatrick, L. Taddei, and A. Sukta. Effects of dose of furosemide on plasma TCO 2 changes in Standardbred horses. J. Equine Vet. Sci. 26: (2006). 7. M.S. Moss. Survey of positive results from racecourse antidoping samples received at Racecourse Security Services Laboratories. Equine Vet. J. 16(1): (1984). 8. L.M. Neto, M.H. Andraus, and M.C. Salvadori. Determination of phenylbutazone and oxyphenylbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography mass spectrometry. J. Chromatogr. B Biomed. Appl. 678: (1996). 9. T.M. Dyke, R.A. Sams, and S.B. Cosgrove. Disposition of flunixin after intramuscular administration of flunixin meglumine to horses. J. Vet. Pharmacol. Ther. 20: (1997). 10. K.W. Hinchcliff, P.S. Morley, and A.J. Guthrie. Efficacy of furosemide for prevention of exercise-induced pulmonary hemorrhage in Thoroughbred racehorses. J. Am. Vet. Med. Assoc. 235(1): (2009). 11. L.R. Soma and C.E. Uboh. Review of furosemide in horse racing: its effects and regulation. J. Vet. Pharmacol. Therap. 21: (1998). 12. K.H. Kline. Milkshakes for the horse. J. Equine Vet. Sci. 24: (2004). 13. C. Luckie, C. Whitney, M. Benoit, L. Taddei, A. Sukta, J. Peterson, D. Schwope, R.E. Gaensslen, and A. Negrusz. Detection and quantification of low levels of benzoylecgonine in equine urine. Forensic Sci. Int. 177: e21 e24 (2008). 14. J. Peterson, M. Benoit, C. Luckie, D. Schwope, A. Sukta, L. Taddei, C. Whitney, and A. Negrusz. A review of etorphine: a toxicological case study. Program and Abstracts, Society of Forensic Toxicologists Annual Meeting, Austin, Texas, S33,