Doping in horses refers to the administration of prohibited substances or methods to enhance performance or alter behavior during competitions. This practice is a concern in equestrian sports due to its potential impact on animal welfare, fair play, and the integrity of the sport. Substances used in doping can range from stimulants and painkillers to sedatives and anti-inflammatory drugs. Detection methods include blood and urine tests, designed to identify the presence of banned substances or their metabolites. This page compiles peer-reviewed research studies and scholarly articles that explore the detection methods, regulatory frameworks, and ethical considerations associated with doping in equine sports.
Lloyd DR, Rose RJ, Duffield AM, Suann CJ.The combination of large doses of sodium bicarbonate and the potent narcotic, etorphine, has reportedly been given to racehorses in attempts to improve their performance and also to "mask" the presence of etorphine in urine samples. The increased urinary output and pH associated with sodium bicarbonate (approximately 500 g) administration may reduce the urinary concentration of etorphine, making it more difficult to detect. Our experiment was designed to examine the effects of this combination. Six Thoroughbred horses were used in a latin-square design with three horse pairs and three treatmen...
González G, Ventura R, Smith AK, de la Torre R, Segura J.A gas chromatographic-mass spectrometric (GC-MS) procedure for the detection of seventeen non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma and urine samples is described. The extraction of the compounds from the biological matrix was performed at acidic pH (2-3) with diethyl ether. Ethereal extracts were washed with a saturated solution of sodium hydrogencarbonate (urine) or treated with a solid mixture of sodium carbonate and sodium hydrogencarbonate (plasma). The ethereal extracts were dried and derivatized by incubation at 60 degrees C with methyl iodide in acetone in the pre...
Bonnaire Y, Dehennin L, Plou P, Toutain PL.A pharmacological dose of a long-acting testosterone ester, testosterone hexahydrobenzoate, was administered intramuscularly to two mares. The time course for some characteristic metabolites in blood and urine was then studied using an analytical method based on gas chromatography-mass spectrometry associated with stable isotope dilution. Among the plasma analytes, testosterone glucuronide was found to be the most adequate indicator for the monitoring of exogenous testosterone up to 2 weeks postadministration if a threshold value of 200 ng/L was applied. In urine, the simultaneous measurement ...
The Journal of nutritionDecember 1, 1994
Volume 124, Issue 12 Suppl 2730S-2735S doi: 10.1093/jn/124.suppl_12.2730S
Snow DH.Since the domestication of the horse and its use in various types of athletic competition, its diet has developed from that associated with grazing to feeding of additional energy in the form of grains up to the present situation when numerous supplements are fed in hopes of enhancing performance. Many if not all of these may be considered as ergogenic aids, and under the rules of racing in many countries should be considered as a prohibited substance. Until recently, a blind eye has been turned to whether the rules should be enforced against many nutritional supplements, for example, vitamins...
Pedroso RC, Salvadori MC, Andraus MH, Lopez NM.A chromatographic method was developed to detect and confirm the presence of succinonitrile (SDN) in horse urine samples, for antidoping control. The urine samples (5 ml) were extracted with diethyl ether and screened by gas chromatography-nitrogen-phosphorus detector and the confirmation of the drug's presence was accomplished by using gas chromatography-mass selective detection. The recovery of extraction was 78 and 81% for 1.0 and 2.0 micrograms ml-1 (relative standard deviation, < 10%), respectively. Urine samples collected after the administration of Energisan were positive for SDN (1-30 ...
Klaus AM, Hapke HJ.Drug treatment of horses which are used in horse-racing is restricted by the regulations of the anti-doping control. Veterinarians and anti-doping control commissions are faced with the problems resulting from the discrepancy between the demand "no drugs in blood/urine of horses at the time of competition" and the need for treatment. The pharmacokinetic data of important antiphlogistics/analgetics (NSAID) for horses given in this article shall facilitate the decision of the veterinarians and commissions whether a horse having been treated with NSAID may participate in a competition or not. Fur...
Pompa G, Caloni F, Montana M, Pasqualucci C.1. Isoxsuprine [1-(4-hydroxyphenyl)-2-(1-methyl-2-phenoxyethylamino)-1- propanol] serum concentrations after single- and multiple-dose administration to horse were investigated using immunoenzymatic ELISA, HPLC-UV and thermospray HPLC-MS methods. 2. Using HPLC-MS, isoxsuprine was detected up to 72 h after a single administration (1.2 mg/kg by gastric probe) and up to 96 h after the end of serial administration (1.2 mg/kg every 12 h for 7 days). 3. ELISA detected the drug up to 96 h after a single dose and up to 6 days after the end of prolonged administration. 4. Isoxsuprine is present in hors...
Stanley S, Wood T, Goodman JP, Henry PA, Woods WE, Chang SL, Tai HH, Watt D, Kwiatkowski S, Blake JW.We have raised antibodies and developed one-step enzyme-linked immunosorbent assays (ELISA) for the diuretics ethacrynic acid and bumetanide as part of a panel of pre- and post-race tests for high potency drugs in racing horses. These ELISA tests are rapid (completed within one hour), sensitive, and can be read by eye. The ELISA detects ethacrynic acid at a drug concentration for half-maximal inhibition (I-50) of about 2.5 ng/mL for the parent drug. After dosing horses intravenously with 5 mg ethacrynic acid per horse, the parent drug or its metabolites are detectable in urine for at least 8 h...
Kamerling SG.Few definitive studies have been performed that unequivocally demonstrate the ability of a drug to alter the performance of a horse. Nonetheless, the use of drugs in competing horses is regulated worldwide. Drugs have been categorized according to their abuse potential. However, there is still some confusion over what is meant by the terms "performance" and "drug." In the racing community, performance means speed, and fatigue and pain are among its greatest detractors. Speed is most appropriately measured on the racetrack. There are a multiplicity of internal and external variables that influe...
Gowen RR, Lengel JG.The control of drug use in performance horses and the policing of rules and regulations to prevent unauthorized drug use are important tasks for agencies overseeing equestrian events. This article describes the roles of the American Horse Shows Association, the Federation Equestre Internationale, and the Association of Racing Commissioners International, Inc, in the policing of drug use in horses competing in events under their control.
Benoit E, Jaussaud P, Besse S, Videmann B, Courtot D, Delatour P, Bonnaire Y.A benzhydrolic metabolite of ketoprofen, formed by reduction of the keto group of the drug, has been identified by gas chromatography-mass spectrometry in equine plasma and urine. After partial synthesis, its structure has been confirmed by UV, IR and 1H NMR spectroscopy. The kinetics of ketoprofen and this metabolite have been monitored in plasma by high-performance liquid chromatography. The two products were quantified in plasma up to 4 and 3 h, respectively, and were detected in urine up to 72 and 24 h, respectively, after a single intravenous administration to horses at the dose of 2.2 mg...
Hagedorn HW, Böck M, Schulz R.Equine blood may contain salicylic acid (SA) taken up as free acid or represents the metabolite of acetylsalicylic acid (ASA). To obtain information of SA in race horses we screened blood samples of trotting-horses routinely drawn to be analyzed for doping substances. The individual values determined followed a Gaussian distribution displaying a geometric mean of 19 ng SA per ml serum. A probit analysis revealed linear relationship (r = 0.995). Additional studies examined the antithrombotic efficacy of ASA in the horse. An oral dose of 300 mg ASA considerably elevated the bleeding time for mor...
Delbeke FT, Debackere M.A gas chromatographic method to measure urinary levels of the central nervous system stimulant fencamfamine and some of its metabolites is described. When 100 mg fencamfamine was given orally to four horses the parent drug could not be detected in the urine. After enzymatic hydrolysis of the urine the major human metabolite, N-desethylated fencamfamine, only accounted for 1% of the dose in 12 h. The major equine metabolites were conjugated parahydroxylated compounds representing 18% of the dose. With regard to horse doping control and analysis, the injudicious use of human doping routine metho...
Hagedorn HW, Schulz R.The use of diuretics in horses subject to doping control is prohibited. Thus, a sensitive screening procedure is required to identify the chemically different diuretics. We communicate here a method to detect three commonly employed acidic diuretics: bumetanide, ethacrynic acid, and furosemide. A liquid-liquid extraction on Extrelut 3 was performed at weak acidic and basic conditions using ethyl acetate as organic solvent. For analysis by GC, the diuretics were methylated on-column in the presence of MSTFA/TMAH, avoiding the commonly employed highly toxic derivatizing agent methyl iodide. For ...
Jaussaud P, Guieu D, Courtot D, Barbier B, Bonnaire Y.A tolfenamic acid metabolite, a hydroxylated product, has been identified in equine plasma and urine samples using gas chromatography-mass spectrometry in the electron-impact and chemical-ionization modes. The method also allows the qualitative monitoring of the elimination of the drug and its metabolites from plasma. The two compounds are detected up to 48 and 24 h, respectively, after a single oral administration of a 30 mg/kg dose. The simultaneous detection of the two products increases the reliability of anti-doping control analysis.
Matassa LC, Woodard D, Leavitt RK, Firby P, Beaumier P.Glycopyrrolate (Robinul) is a quaternary ammonium salt which serves as a respiratory enhancing drug. It is reportedly used in horse racing to improve breathing. Extraction of glycopyrrolate from equine urine employing unique solid-phase extraction techniques gave a residue suitable for liquid chromatography-tandem mass spectrometry (LC-MS-MS) and gas chromatography-mass spectrometry (GC-MS). LC-MS-MS analysis employed an extract derived from 5 ml of urine subjected to cation-exchange chromatography. The daughter ion of m/z 318 monitored in the positive-ion mode was m/z 116. Recovery of glycopy...
Stanley S, Jeganathan A, Wood T, Henry P, Turner S, Woods WE, Green M, Tai HH, Watt D, Blake J.We have raised antibodies to morphine and etorphine and developed one-step enzyme-linked immunosorbent assays (ELISA) for these drugs as part of a panel of post race tests for drugs in racing horses. These tests are simple, can be completed in 2 h, and can be read by visual inspection. The morphine ELISA has an I50 for morphine of about 1.5 ng/mL, while the etorphine ELISA has an I50 for etorphine of 250 pg/mL. Cross-reactivity studies show that the antimorphine antibody cross-reacts well with levorphanol, hydromorphone, and oxycodone, while the anti-etorphine antibody showed no cross-reactivi...
Teale P, Houghton E.A screening procedure for anabolic steroid residues in horse urine has been developed based upon solid-phase extraction and gas chromatographic/mass spectrometric analysis in the selected ion mode. For moderate sample throughput the method provides a viable alternative to radioimmunoassay screening and has advantages over the latter technique due to its flexibility, specificity and ability to detect a number of steroids in a single analysis. Full automation of the gas chromatographic/mass spectrometric analysis is an additional feature of the methodology.
Bourdon R.Toxicomania and doping give rise to an increasing number of drug measurements in the body fluids. Consequently the analysts have to face, at one and the same time, ethical and analytical problems. Should the analyst participate to investigations organised in order to disclose a toxicomania in a working place? The author suggests a positive answer as long as the adduct person benefits to a social and medical care. What is the analytical meaning of a positive test? Taking into account the increasing sensitivity of the methods used, thresholds have to be established, at least for the blood concen...
Edlund O, Bowers L, Henion J, Covey TR.Urine samples were spiked with [17-methyl-2H3]methandrostenolone as internal standard and extracted with a mixture of dichloromethane and cyclohexane. The organic phase was concentrated and injected onto a short octyl-silica column (30 mm x 4.6 mm I.D.) for separation of methandrostenolone and 17-epimethandrostenolone. The effluent from the column was connected to a Sciex TAGA 6000E triple quadrupole mass spectrometer equipped with an atmospheric pressure ion source for sampling of ions generated by a heated pneumatic nebulizer with corona discharge ionization. This ion source produced abundan...
Wood T, Tai CL, Taylor DG, Woods WE, Wang CJ, Houtz PK, Tai HH, Weckman TJ, Yang JM, Sturma L.Detomidine is a potent non-narcotic sedative agent which is currently in the process of being approved for veterinary clinical use in the United States. Since no effective screening method in horses is available for detomidine, we have developed an 125I radioimmunoassay for detomidine in equine blood and urine as part of a panel of tests for illegal drugs in performance horses. Our 125I radioimmunoassay has an I-50 for detomidine of approximately 2 ng/ml. Our assay shows limited cross-reactivity with the pharmacodynamically similar xylazine, but does not cross-react with acepromazine, epinephr...
Kwiatkowski S, Sturma L, Dai MR, Tai HH, Watt DS, Tai CL, Woods WE, Weckman TJ, Yang JM, Wood T.We have developed and evaluated a one step enzyme-linked immunosorbent assay (ELISA) test and a particle concentration fluorescence immunoassay (PCFIA) test for acepromazine as part of a panel of pre- and post-race tests for illegal medications in racing horses. These tests are rapid, sensitive and economical and development of the tests occurred in less than seven months. The ELISA test detects acepromazine with an I-50 of about 150 pg/ml. In vivo, it readily detects the presence of acepromazine or its metabolites in equine blood and urine from 8 to 72 hours or longer, respectively, after adm...
Appolonova SA, Baranov PA, Mesonzhnik NV, Brazhnikova DO, Rodchenkov GM.A method is described for the determination of mesocarb abuse in equestrian sport by combining gradient liquid chromatography and electrospray ionization tandem mass spectrometry. Mesocarb was administrated orally to two horses at a dose of 50 µg/kg. Urine samples were collected up to 120 h post administration. Hydrolyzed and conjugated urine fractions were handled using liquid-liquid extraction (LLE). The identity of the parent drug and metabolites was confirmed using liquid chromatography combined with tandem mass spectrometry (MS/MS). Mesocarb and seven metabolites were detected in horse...
Hertzsch R, Emmerich IU, Lachenmeier DW, Sproll C, Monakhova YB, Aboling S, Bachmann U, Vervuert I.Opioid alkaloids were identified in the urine of horses during an anti-doping control and in a case of intoxication. In both cases, it was suspected that the horses had ingested poppy-contaminated feed. To verify this suspicion, possible opioid alkaloid sources in Germany were identified through a literature research. Additionally, the contaminated feed was botanically and chemically analysed. The results indicated that both cases were most probably caused by the poppy in the feed. This highlights the previously underestimated risk of an intake of poppy-contaminated feed in horses. Recommendat...
Van Eenoo P, Delbeke FT, Deprez P.Clenbuterol, a beta-adrenergic agonist, is used in the treatment of recurrent airway obstruction in horses. It is prohibited by horse racing authorities, because of its stimulating and growth-promoting properties. However, information on detection times of clenbuterol after administration by nebulization is lacking. In this study, a fast, sensitive quantitative GC-MS(2) method for the detection of clenbuterol in urine was developed. Alkaline liquid-liquid extraction was followed by derivatization to a cyclic methyl boronate derivative and analysis on a Finnigan MAT GCQ instrument. Method valid...
Wenzel R, Major D, Hesp K, Doble P.Regulating authorities in the racing industry have restricted the administration of potentially performance enhancing cobalt salts to horses. There are severe penalties for trainers presenting horses with elevated urine cobalt concentrations, and compliance is ensured via analysis of total urinary cobalt at thresholds of 100 μg/L. When cobalt is present as part of the cobalamin molecule it is not considered performance enhancing. This paper demonstrates that a horse can excrete a significant proportion of a commercially available vitamin B12 injection in urine without metabolic modification...
Guan F, Uboh CE, Soma LR, You Y, Liu Y, Li X.Androgenic and anabolic steroids (AASs) are a class of chemical substances closely related to testosterone in molecular structure. They can be abused to enhance performances in human and equine athletes, and are banned by the sports authorities. To assist with method development for doping analyses of AASs, investigations were conducted to correlate their product ion profiles with the molecular structures. Although very similar in chemical structure, AASs generated noticeably different product ion profiles from collision-induced dissociation (CID). On the basis of both outlines of the product ...
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.In equine sport, salicylic acid is prohibited with a threshold level of 750 microg mL(-1) in urine; hence, doping control laboratories have to establish quantitative and qualitative methods for its determination. A simple and rapid liquid chromatographic/mass spectrometric method was developed and validated for the quantification and identification of salicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the liquid chromatography/quadrupole time-of-flight mass spectrometry system. Electrospray ionization i...
Costello S, Heffron B, Taddei L, Benoit M, Hurt L, Simpson L, Bishop J, Folker-Calderon D, Negrusz A.Fluphenazine, a potent antipsychotic used to treat schizophrenia in humans, is used in racehorses as a performance-enhancing drug, and for that reason it has been banned by the Association of Racing Commissioners International. A liquid chromatography-tandem mass spectrometry method for detecting and quantitating fluphenazine in equine serum was developed and validated. The method was then employed to quantitate fluphenazine in serum samples collected from three study horses after intramuscular injection of fluphenazine decanoate. Stability testing showed that fluphenazine is stable in unextra...
Nakajima T, Matsumoto T.Horse racing in Japan consists of two systems, the National (10 racecourses) and the Regional public racing (32 racecourses) having about 2,500 racing meetings in total per year. Urine or saliva samples for dope testing are collected by the officials from thw winner, second and third, and transported to the laboratory in a frozen state. In 1975, 76, 117 samples were analyzed by this laboratory. The laboratory provides the following four methods of analysis, which are variously combined by request. (1) Method for detection of drugs extracted by chloroform from alkalinized sample. (2) Methods fo...
Aramaki S, Ishidaka O, Suzuki E, Momose A, Umemura K.In a doping test for racing horses, it is useful for the elucidation of the illegal use of drugs if one can estimate the time at which the detected drug was administered. In order to estimate the time which has elapsed after the administration of caffeine (CA) into horses, the ratios of concentration for the respective metabolites to the unchanged CA in the plasma or the urine were determined. These ratios have been known to be independent of the dose of CA. The relationship between [plasma or urinary concentration of a metabolite]/ [plasma or urinary concentration of the unchanged drug] and t...
So YM, Wong JKY, Wong ASY, Tse ATL, Wan TSM, Ho ENM.The erythropoietin mimetic peptide 1 linear form (EMP1-linear), GGTYSCHFGPLTWVCKPQGG-NH , was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials using ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The white crystalline powder, allegedly used for doping racehorses, was found to contain around 2% (w/w) of EMP1-linear. EMP1-linear can be cyclised in equine plasma at physiological temperature of 37°C by forming an intramolecular disulfide bond to give EMP1, which is a well-known erythropoiesis st...
Kwiatkowski S, Sturma L, Dai MR, Tai HH, Watt DS, Tai CL, Woods WE, Weckman TJ, Yang JM, Wood T.We have developed and evaluated a one step enzyme-linked immunosorbent assay (ELISA) test and a particle concentration fluorescence immunoassay (PCFIA) test for acepromazine as part of a panel of pre- and post-race tests for illegal medications in racing horses. These tests are rapid, sensitive and economical and development of the tests occurred in less than seven months. The ELISA test detects acepromazine with an I-50 of about 150 pg/ml. In vivo, it readily detects the presence of acepromazine or its metabolites in equine blood and urine from 8 to 72 hours or longer, respectively, after adm...
Ungemach FR.Doping in performance horses is defined as the "illegal application of any substance, except normal diet, that might modify the natural and present capacities of the horse at the time of the race." The prohibition of doping is mainly based on the protection of animals. Doping can be performed with various aims: "doping to win" can be regarded as the classical method by mobilization of overphysiological capacities. Such positive doping may be classified as an acute form using psychomotoric stimulants, as a chronic form using anabolic hormones, and as a paradoxical form using small doses of neur...
Dehennin L, Bonnaire Y, Plou P.The dietary supplements 19-norandrostenedione and 19-norandrostenediol are potential metabolic precursors of nandrolone. They are considered by law in the United States as prohormones without proven therapeutic, curative or diagnostic properties, and therefore available as over-the-counter drugs. Oral dosages of 0.1-1 mg/kg body weight were readily absorbed in the equine intestinal tract and thereby led to urinary excretion of drastically increased 5alpha-estrane-3beta,17alpha-diol conjugates, which are known to be final metabolites of nandrolone. The actual rules for detection of illicit nand...
Decloedt AI, Bailly-Chouriberry L, Vanden Bussche J, Garcia P, Popot MA, Bonnaire Y, Vanhaecke L.To ensure fair competition and to protect the horse's welfare, horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, regulatory authorities list all substances that are not allowed in competition, including most anabolic-androgenic steroids. As zero-tolerance is retained, the question arose whether the consumption of mouldy feed could lead to the excretion of steroids, due to the biotransformation of plant phytosterols to steroids. A rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analyt...
Lloyd DR, Rose RJ, Duffield AM, Suann CJ.The combination of large doses of sodium bicarbonate and the potent narcotic, etorphine, has reportedly been given to racehorses in attempts to improve their performance and also to "mask" the presence of etorphine in urine samples. The increased urinary output and pH associated with sodium bicarbonate (approximately 500 g) administration may reduce the urinary concentration of etorphine, making it more difficult to detect. Our experiment was designed to examine the effects of this combination. Six Thoroughbred horses were used in a latin-square design with three horse pairs and three treatmen...
Wang CC, Hartmann-Fischbach P, Krueger TR, Wells TL, Simonson A, Compton JC.Due to opiorphin's analgesic and antidepressant functions, its illicit use is rumored in some racing jurisdictions. Opiorphin is very difficult to detect due to its hydrophilic nature and rapid degradation in plasma and urine samples. METHODOLOGY & RESULTS: We have developed a sensitive, reliable method for opiorphin detection and confirmation in equine samples, using EDTA to inhibit analyte degradation between the time of collection and analysis. Opiorphin was extracted by weak cation exchange followed by analysis using HILIC-MS/MS. The method was validated and the LOD was determined to b...
Wong JKY, Kwok WH, Chan GHM, Choi TLS, Ho ENM, Jaubert M, Bailly-Chouriberry L, Bonnaire Y, Cawley A, Ming Williams H, Keledjian J, Brooks L....Acadesine, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control del...
Liu Y, Uboh CE, Soma LR, Li X, Guan F, You Y, Rudy JA, Chen JW.Gabapentin (GPT) is an antiepileptic drug that was approved in 1993 for use in the management of neurotrophic pain and as an adjunctive therapy for refractory partial seizure in humans. It is also being tested in veterinary medicine as an adjunctive medication in the treatment of pain due to laminitis, neuropathic, or chronic pain. Gabapentin is readily available by prescription and even on the internet; therefore, it has the potential of being used in racehorses to mask pain. It is for this reason that a sensitive liquid chromatography-tandem mass spectrometry method has now been developed fo...
Machnik M, Gerlach M, Kietzmann M, Niedorf F, Thevis M, Schenk I, Guddat S, Düe M, Schänzer W.The anti-doping rules of national and international sport federations ban any use of tetrahydrogestrinone (THG) in human as well as in horse sports. Initiated by the THG doping scandals in human sports a method for the detection of 3-keto-4,9,11-triene steroids in horse blood and urine was developed. The method comprises the isolation of the analytes by a combination of solid phase and liquid-liquid extraction after hydrolysis and solvolysis of the steroid conjugates. The concentrations of THG in blood and urine samples were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS)...
Kusano K, Minamijima Y, Mashita S, Kunii H, Yamashita S, Nagata S.Repeated topical application of indomethacin is common in Japanese racehorses, despite the lack of pharmacokinetic data. Objective: To determine the concentrations of indomethacin and its metabolite, desmethylindomethacin, in plasma and urine of Thoroughbreds topically treated repeatedly with indomethacin. Methods: In vivo experimental. Methods: Seven female Thoroughbreds were topically treated with 50 g of 1% indomethacin cream per horse to the back and hips (500 mg of indomethacin/head/2400 cm , 0.21 g/cm ) for 3 consecutive days. Samples were pretreated by protein precipitation for plas...
Kurosawa M, Nagata S, Takeda F, Mima K, Hiraga A, Kai M, Taya K.Our aim was to investigate plasma catecholamine responses to so-called 'doping' drugs and exercise in Thoroughbreds. Plasma adrenaline (Ad) and noradrenaline (NA) were determined after the administration of caffeine and promazine hydrochloride (PRZ) using a high performance liquid chromatographic method. Caffeine or PRZ was administered i.m. to Thoroughbreds and its effects on plasma catecholamines at rest and during exercise were compared with the saline control. The treadmill exercise was performed 1 h after administration. A dose of 5.0 mg/kg bwt caffeine was found to significantly increase...
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.In equine sport, theobromine is prohibited with a threshold level of 2 microg mL(-1) in urine, hence doping control laboratories have to establish quantitative and qualitative methods for its determination. Two simple liquid chromatography/mass spectrometry (LC/MS) methods for the identification and quantification of theobromine were developed and validated using the same sample preparation procedure but different mass spectrometric systems: ion trap mass spectrometry (ITMS) and time-of-flight mass spectrometry (TOFMS). Particle-free diluted urine samples were directly injected into the LC/MS ...
Viljanto M, Cutler C, Taylor P, Habershon-Butcher J, Gray B.MK-0677 (ibutamoren) is an orally active non-peptide growth hormone secretagogue that binds to the ghrelin receptor stimulating the secretion of endogenous growth hormone. It is one of the most prevalent performance-enhancing compounds currently available online and is potentially subject to abuse both in human and equine sports. The aim of the current study was to investigate whether it could be detected in equine hair following oral administration of MK-0677 mesylate to a Thoroughbred racehorse. MK-0677 and its O-dealkylated metabolite were extracted using an existing method for prohibited s...
Schenk I, Broussou D, Roques B, Lagershausen H, Machnik M, Röttgen H, Toutain PL, Thevis M.The combination of sulfadoxine (SDO) with trimethoprim (TMP) is widely used in veterinarian medicine. The aim of the present study was to compare excretion profiles and detection time windows of SDO and TMP in plasma and urine by means of a validated quantitative method. Eight horses received a single intravenous (i.v.) dose of 2.7 mg TMP and 13.4 mg SDO per kg bodyweight. Plasma and urine samples were collected up to 15 and 70 days post-administration, respectively. While urine samples underwent an enzymatic hydrolysis, plasma samples were proteolysed before further analysis. After solid-...
Ho EN, Kwok WH, Wong AS, Wan TS.Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) m...
Hagedorn HW, Meiser H, Zankl H, Schulz R.The misuse of opiates in racehorses relates to their effect of increasing locomotor activity. Because methadone, a narcotic analgesic, has been suspected of use as a doping compound in the past, it was added to the list of banned drugs and should be considered in doping control. Because the literature fails to provide information on detection of methadone in blood or urine of horses, an enzyme-linked immunosorbent assay was developed to monitor this narcotic in equine body fluids. Combined with high-performance liquid chromatography, the immunoassay also served to confirm positives indicated b...
Stanley S, Jeganathan A, Wood T, Henry P, Turner S, Woods WE, Green M, Tai HH, Watt D, Blake J.We have raised antibodies to morphine and etorphine and developed one-step enzyme-linked immunosorbent assays (ELISA) for these drugs as part of a panel of post race tests for drugs in racing horses. These tests are simple, can be completed in 2 h, and can be read by visual inspection. The morphine ELISA has an I50 for morphine of about 1.5 ng/mL, while the etorphine ELISA has an I50 for etorphine of 250 pg/mL. Cross-reactivity studies show that the antimorphine antibody cross-reacts well with levorphanol, hydromorphone, and oxycodone, while the anti-etorphine antibody showed no cross-reactivi...
Decloedt A, Bailly-Chouriberry L, Vanden Bussche J, Garcia P, Popot MA, Bonnaire Y, Vanhaecke L.Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to...
Hagedorn HW, Meiser H, Zankl H, Schulz R.Due to its tranquilizing properties, the tricyclic antidepressant doxepin may be misused as a doping agent in competition horses. Therefore, efficient analytical procedures are required to detect this drug in samples submitted for doping control. To screen for parent doxepin in equine blood and urine, a less specific method has been accepted employing gas chromatography (GC) combined with electron impact (EI) mass spectrometry (MS). The aim of this study was identification of doxepin metabolites providing more specific MS data to verify positives resulting from screening. Thus, after a horse w...
Ho EN, Kwok WH, Leung DK, Wan TS, Wong AS.Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incub...
