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.
Takeda A, Tanaka H, Shinohara T, Ohtake I.A sample preparation method for mass chromatographic detection of doping drugs from horse plasma is described. Bond Elut Certify (1 g/6 ml) is used for the extraction of 4 ml of horse plasma. Fractionation is performed with 6 ml of CHCl3-Me2CO (8:2) and 5 ml of 1% TEA-MeOH according to its property. Simple and effective clean-up based on non-aqueous partitioning is adopted to remove co-eluted contaminants in both acid and basic fractions. Two kinds of 1-(N,N-diisopropylamino)-n-alkanes are co-injected with the sample into the GC-MS system for the calculation of the retention index. Total recov...
de Kock SS, Rodgers JP, Swanepoel BC.Previous studies have shown that insulin-like growth factor 1 (IGF-1) is a promising marker for the detection of growth hormone (GH) abuse in the horse. The significant increases observed with GH administration in comparison to natural levels imply the possibility of setting a threshold level for IGF-1 that would be indicative of GH abuse. Although an immunoradiometric assay (IRMA) has been identified as a reliable screening method, a more specific IGF-1 quantification method needs to be developed for the prosecution of GH abuse by horseracing authorities. This study describes such an HPLC ele...
Lehner AF, Harkins JD, Karpiesiuk W, Woods WE, Robinson NE, Dirikolu L, Fisher M, Tobin T.Clenbuterol is a beta2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivoca...
Dirikolu L, Lehner AF, Karpiesiuk W, Harkins JD, Woods WE, Carter WG, Boyles J, Fisher M, Tobin T.Lidocaine is a local anesthetic drug that is widely used in equine medicine. It has the advantage of giving good local anesthesia and a longer duration of action than procaine. Although approved for use in horses in training by the American Association of Equine Practitioners (AAEP), lidocaine is also an Association of Racing Commissioners International (ARCI) Class 2 drug and its detection in forensic samples can result in significant penalties. Lidocaine was observed as a monoprotonated ion at m/z 235 by ESI+ MS/MS (electrospray ionization-positive ion mode) analysis. The base peak ion at m/...
Harkins JD, Karpiesiuk W, Tobin T, Dirikolu L, Lehner AF.Ropivacaine is a local anesthetic that has a high potential for abuse in racing horses. It can be recovered from urine collected after administration as a hydroxylated metabolite following beta-glucuronidase treatment of the urine. Based on these findings, it has been inferred that ropivacaine is present in equine urine as a glucuronide metabolite; however, these metabolites have never been directly identified. Using ESI+/MS/MS, the presence of a [M+H]+ molecular ion of m/z 467 was demonstrated in urine corresponding to the calculated mass of a hydroxyropivacaine glucuronide +1. The abundance ...
Grippa E, Santini L, Castellano G, Gatto MT, Leone MG, Saso L.Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite ...
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...
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...
Harkins JD, Lehner A, Karpiesiuk W, Woods WE, Dirikolu L, Boyles J, Carter WG, Tobin T.Bupivacaine is a potent local anaesthetic used in equine medicine. It is also classified as a Class 2 foreign substance by the Association of Racing Commissioners International (ARCI). The identification of residues in postrace urine samples may cause regulators to impose significant penalties. Therefore, an analytical/pharmacological database was developed for this medication. The highest no-effect dose (HNED) for the local anaesthetic effect of bupivacaine was determined to be 0.25 mg by using an abaxial sesamoid local anaesthetic model. Administration of the HNED of bupivacaine to eight hor...
Garbís SD, Hanley L, Kalita S.Thiazide-based diuretics are included in the list of banned drugs in the horse-racing industry. One effect of their misuse is increased urine flow, contributing to dilution of other doping agents. Their determination is essential in ensuring compliance to horse-racing regulation. This study evaluates the feasibility of using liquid chromatography/mass spectrometry (LC/MS) with electrospray and atmospheric pressure chemical ionization interfaces to analyze thiazidic diuretics in equine urine samples. Existing LC and gas chromatography/MS methods are limited in their applicability to thiazide an...
Chua HC, Stewart B, Lim BH, Lee HK.A chromatographic method was developed to detect and confirm the presence of chlorpropamide (I) in horse plasma samples, for antidoping control. The plasma sample (1 ml) was extracted with dichloromethane and screened by high-performance liquid chromatography, and confirmation of the drug's presence was accomplished by using gas chromatography-mass spectrometry (GC-MS). The limit of detection was found to be 3.5 ng/ml at a signal-to-noise ratio of three. Derivatization of I with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane allowed for highly stable, accurate and sen...
Laidler P, Cowan DA, Houghton E, Kicman AT, Marshall DE.Recognition by the legal authorities that growth hormones (GHs) may be abused to improve sporting performance and/or physique has led to the implementation of controls that make it an offence to produce, supply, possess or import and export GHs, with intent to supply, without the authority to do so. A method is described for the discriminatory analysis of human, equine, porcine and bovine GHs for forensic purposes. Peptide-mass mapping by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry following tryptic digestion gave sequence coverages of 97.4%, 93.7...
Wilsdorf G, Graf G.Since the early twenties several institutes of the Veterinary College in Berlin were involved in the development of microchemical analyzing methods for doping-alkaloids and clinical studies in doped race horse. This research made it possible to build national acting structures against the abuse of doping in race horses, which soon became a model for similar activities in other countries, for instance Greece, Japan and the United States. Concerning the results of their research, the scientists of the Veterinary College in Berlin have got an enormous part in fighting against doping in race horse...
Soma LR, Uboh CE.Furosemide has been used empirically and has been legally approved for many years by the US racing industry for the control of exercise-induced pulmonary haemorrhage (EIPH) or bleeding. Its use in horses for this purpose is highly controversial and has been criticized by organizations outside and inside of the racing industry. This review concentrates on its renal and extra-renal actions and the possible relationship of these actions to the modification of EIPH and changes in performance of horses. The existing literature references suggest that furosemide has the potential of increasing perfo...
Short CR, Sams RA, Soma LR, Tobin T.The primary reason for developing the ARCI Uniform Classification of Foreign Substances was to give stewards and other racing regulators guidelines to assist them in understanding the relative performance effects and general offensiveness to the Rules of Racing of various drugs and medications. As such, these guidelines have been very useful in the world of racing regulation--officially or unofficially--because this classification system, for the first time, places a relative number on the inappropriateness of any one of more than 750 agents appearing in forensic samples taken from racing hors...
Düe M.A survey is given about the situation of "doping" of horses. A definition of the term "doping" is delivered which is up to date and considers the matters of animal welfare. Existing regulations will be discussed in view of recent results of research. Different regulations for horses as well as humans in sports will be compared. Also different attempts of solutions for the regulatory body will be presented, which may change the regulatory significance. Finally a preview about actual questions related to doping is given.
Andraus MH, Siqueira ME.An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC-MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC-MS system. The ELISA test (20 microl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC-MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC-MS method in the SIM mode using nalbup...
Aguilera R, Becchi M, Mateus L, Popot MA, Bonnaire Y, Casabianca H, Hatton CK.A gas chromatography-combustion-isotope ratio mass spectrometry method for confirmation of hydrocortisone abuse in horseracing and equine sports is proposed. Urinary hydrocortisone was converted to a bismethylenedioxy derivative which presents good gas chromatographic properties and brings an extra carbon contribution of only two carbon atoms. Synthetic hydrocortisone has a different 13C abundance from that of natural urinary horse hydrocortisone and the difference is significant, therefore exogenous and endogenous hydrocortisone can be distinguished.
Uehara N, Momose A.The doping test method used in a horse race requires the accurate detection of a wide variety of drugs and metabolites as well as the rapidity in order to examine a large number of samples within a limited time. For this purpose, the routine method consists of a preliminary screening and a confirmatory test. In this paper, a historical review for the development of the doping test method in Japan is described. The metabolism and pharmacology of drugs in horses are also discussed.
Ribeiro Neto LM, Spinosa HS, Salvadori MC.Dexamethasone is a corticosteroid drug widely used in racehorses because of its anti-inflammatory effect. It is, therefore, frequently detected in antidoping tests. A method for the antidoping control of dexamethasone in equine urine using screening by ELISA and confirmation by immunoaffinity chromatography combined with reversed-phase high-performance liquid chromatography-diode array detection (HPLC-DAD) is described. The ELISA test is frequently used in antidoping tests for its sensitivity, relative speed, and low cost. The test showed linearity in the range of 4-500 ng/mL of urine, and the...
Klaus AM, Schlingloff Y, Kleinitz U, Böttcher M, Hapke HJ.The pharmacokinetic behaviour of dipyrone metabolite 4-MAA in serum was determined in seven horses of different breeds after a single intravenous dose administration. A biexponential formula was fitted to the serum concentration vs. time data. The median half-life of the elimination phase (t1/2 beta) was 4.85 h (range 5.04 h), the median volume of distribution (Vd(area)) was 1.85 L/kg (range 3.2 L/kg) and median of total clearance was 4.0 mL/min/kg (range 2.3 mL/min/kg).
Popot MA, Houghton E, Ginn A, Jones M, Teale P, Samuels T, Lassourd V, Dunnett N, Cowan DA, Bonnaire Y, Toutain PL.The purpose of the present report was to estimate the population parameters of cortisol concentrations in urine, an endogenous hormone used as a 'doping' agent and for which an international threshold (1.0 micrograms/ml) has been proposed. Two data bases (French and UK) corresponding to 112 and 142 samples, respectively were considered. Urine was collected under specific post competition conditions. Cortisol concentrations were obtained by validated methods (HPLC for the French samples, and GC-MS for UK samples). No difference was observed between the 2 data sets and statistical analyses were ...
Klaus AM, Hapke HJ.This review compromises data about endogenous cortisol and its physiological variations in horses. The influence of synthetic glucocorticoids on the endogenous cortisol concentrations is discussed as well. The second part of the review summarizes detection times of therapeutically used glucocorticoids (dexamethasone, betamethasone, triamcinolone, prednisone, prednisolone, methylprednisolone and hydrocortisone) in the horse and their implication for doping control.
Souillard A, Audran M, Bressolle F, Jaussaud P, Gareau R.The pharmacokinetics of recombinant human Epo (rHuEpo) were investigated after subcutaneous administration to horses. Four horses received a single 30IU kg-1 dose of rHuEpo. One horse received three repeated doses of 120 IU kg-1 at 48 h intervals. Plasma erythropoietin (Epo) was measured by radioimmunoassay. In both cases pharmacokinetic parameters were evaluated using a one-compartment open model and first-order input and output rates. The mean values (+/-SD) for elimination half-life, CL/F, and Vd/F after a single dose were 12.9 +/- 3.34 h, 11.8 +/- 4.96 L h-1, and 233 +/- 126 L, respectivel...
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...
Neto LM, Andraus MH, Salvadori MC.A method is described for the qualitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deprotein...
Hagedorn HW, Zuck S, Schulz R.The azaphenothiazine neuroleptic prothipendyl (Dominal) is suspected to be administered illegally at low doses to race-horses to improve their performance. Since for this species pharmacokinetic data of the drug are missing we studied its elimination from blood and urine in a standard-bred mare. At a low (subtherapeutic) dose (i.v., 0.24 mg/kg) the horse is described to be less excited while locomotor activity and attention remain unaffected. In contrast, sedation and ataxia are brought about at 1 mg/kg (therapeutic dose). Identification of prothipendyl given i.v. at subtherapeutic doses was a...
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...
Pu F, McKinney AR, Stenhouse AM, Suann CJ, McLeod MD.A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabo...
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...
Cartoni GP, Montanaro M.The results and the improvement of the analytical procedures adopted for the control of doping in horses will be reported. This control has been systematically carried out in Italy for about 10 years in the laboratories of Italian Federation of Sport and Medicine in which the biological samples for the control of doping in various sport activities (football, cycling, athletics etc.) are also examined. In this way it is possible to use the same instruments for all these similar problems and compare the results. The analytical procedure is based on the following steps: 1) Extraction of the sampl...
Van Eenoo P, Delbeke FT, Roels K, Baert K.Non-steroidal anti-inflammatory drugs (NSAIDs) are prohibited by the International Federation of Horse Racing Authorities but are commonly used in veterinary practice. Plasma and urinary concentrations of the NSAID tolmetin were determined by a high-performance liquid chromatographic procedure with UV detection following oral administration of a dose of 1 g to six fasted untrained standard bred mares. With a limit of quantitation (LOQ) of 0.05 microg/ml tolmetin was present in plasma for 9-12 h post-administration. Maximum concentrations of 2.1+/-0.89 microg/ml were found after 0.7+/-0.25 h. T...
Kim Y, Seo C, Oh S, Kwak J, Jung S, Sin E, Kim H, Ji M, Lee HS, Park HJ, Lee G, Yu J, Kim M, Lee W, Paik MJ.Loxoprofen is a non-steroidal anti-inflammatory drug of the 2-arylpropionic acid type, which has used to treat musculoskeletal disorders in the horse racing industry. However, it has also used illicitly to mask clinical signs of inflammation and pain in racehorses. Thus, its accurate analysis has become an important issue in horse doping laboratories. In this study, an analytical method of loxoprofen was developed as tert-butyldimethylsilyl (TBDMS) derivative by gas chromatography-mass spectrometry (GC-MS). Characteristic fragment ions of [M-15], [M-57], and [M-139] permitted the accurate and ...
Woods WE, Chay S, Houston T, Blake JW, Tobin T.The efficacy of testing for illegal drugs in race horses was surveyed by evaluating 27 questionnaires received from 28 racing jurisdictions polled. Large variations in the number of samples tested and drugs detected were reported. Some jurisdictions reported only illegal medications, whereas others also reported permitted medications. To facilitate comparison, stimulants, depressants, local anesthetics, narcotic analgesics, and tranquilizers were classified as hard drugs. Other drugs, which are legal in some jurisdictions, were classified as soft. To evaluate the efficacy of testing, positive ...
Hülsemann F, Flenker U, Machnik M, Schänzer W.Sodium bicarbonate administration to horses prior to competition in order to enhance the buffer capacity of the organism is considered as a doping offence. The analysis of the isotopic composition of urinary bicarbonate/CO(2) (TCO(2)) may help to identify an exogenous bicarbonate source, as technical sodium bicarbonate exhibits elevated delta(13)C values compared with urinary total carbon. The isolation of TCO(2) from 60 equine urine samples as BaCO(3) followed by an isotopic analysis shows a significant variability of delta(13)C for TCO(2) of more than 10 per thousand. The delta(13)C of total...
Jaussaud P, Courtot D, Doron P, Guyot JL.Chronopharmacokinetics of intravenous phenylbutazone in the horse was studied with the aim of antidoping control. Among parameters studied, the single one which seemed to depend on circadian rhythm was the elapsed time between the injection and the plasmatic peak. There was no relationship between the injection time and the both parameters: half-life and time required to reach the forensic level of 4 micrograms/ml. This later, and oxyphenbutazone/phenylbutazone ratio, should depend on individual factors. Therefore, the injection time should not be a main parameter for the phenylbutazone evalua...
Leung DKK, Wong ASY, Zhou QL, Wan TSM, Ho ENM.A non-target variable Data Independent Acquisition (vDIA) workflow based on accurate mass measurements using a Q Exactive OrbiTrap is presented for the first time for equine doping control testing. The vDIA workflow uses a combination of MS1 events (1 to 2) and multiple vDIA events to cover the analytes of interest. The workflow basically captures a digital image of a sample allowing all relevant MS1 and MS2 data to be recorded. In theory, the workflow can accommodate an unlimited number of analytes as long as they are amenable to the sample extraction protocol and fall within the mass limits ...
Knych HK, Stanley SD, Benson D, Arthur RM.Guaifenesin is an expectorant commonly used in performance horses to aid in the clearance of mucus from the airways. Guaifenesin is also a centrally acting skeletal muscle relaxant and as such is a prohibited drug with withdrawal necessary prior to competition. To the authors' knowledge, there are no reports in the literature describing single or multiple oral administrations of guaifenesin in the horse to determine a regulatory threshold and related withdrawal time. Therefore, the objective of the current study was to describe the pharmacokinetics of guaifenesin following oral administration ...
Guan F, Uboh CE, Soma LR, Luo Y, Jahr JS, Driessen B.Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human pl...
Levina V, Timms M, Vine J, Steel R.A synthetic Interleukin-1 receptor antagonist peptide with the sequence Acetyl-Phe-Glu-Trp-Thr-Pro-Gly-Tyr-Trp-Gln-Pro-Tyr-Ala-Leu-Pro-Leu-OH has been identified in a vial seized during a stable inspection. The use of peptide-based Interleukin-1 receptor antagonists as anti-inflammatory agents has not been previously reported, making this peptide the first in a new class of sports doping peptides. The peptide has been characterized by high-resolution mass spectrometry and a detection method developed based on solid-phase extraction and liquid chromatography - triple quadrupole mass spectrometr...
Takeda A, Tanaka H, Shinohara T, Ohtake I.A sample preparation method for mass chromatographic detection of doping drugs from horse plasma is described. Bond Elut Certify (1 g/6 ml) is used for the extraction of 4 ml of horse plasma. Fractionation is performed with 6 ml of CHCl3-Me2CO (8:2) and 5 ml of 1% TEA-MeOH according to its property. Simple and effective clean-up based on non-aqueous partitioning is adopted to remove co-eluted contaminants in both acid and basic fractions. Two kinds of 1-(N,N-diisopropylamino)-n-alkanes are co-injected with the sample into the GC-MS system for the calculation of the retention index. Total recov...
Kwok KY, Chan GHM, Kwok WH, Wong JKY, Wan TSM.Selective estrogen receptor modulators (SERMs) are chemicals that possess the anti-oestrogenic activities that are banned 'in' and 'out' of competition by the World Anti-Doping Agency (WADA) in human sports, and by the International Federation of Horseracing Authorities (IFHA) in horseracing. SERMs can be used as performance-enhancing drugs to boost the level of androgens or to compensate for the adverse effects as a result of extensive use of androgenic anabolic steroids (AASs). SERMs have indeed been abused in human sports; hence, a similar threat can be envisaged in horseracing. Numerous an...
Chua HC, Stewart B, Lim BH, Lee HK.A chromatographic method was developed to detect and confirm the presence of chlorpropamide (I) in horse plasma samples, for antidoping control. The plasma sample (1 ml) was extracted with dichloromethane and screened by high-performance liquid chromatography, and confirmation of the drug's presence was accomplished by using gas chromatography-mass spectrometry (GC-MS). The limit of detection was found to be 3.5 ng/ml at a signal-to-noise ratio of three. Derivatization of I with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane allowed for highly stable, accurate and sen...
Wong ASY, Choi TLS, Kwok KY, Wong JKY, Wan TSM, Ho ENM.Antipsychotics are banned substances and considered by the Fédération Equestrian Internationale (FEI) to have no legitimate use in equine medicine and/or have a high potential for abuse. These substances are also prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). Over the years, antipsychotics have been abused or misused in equestrian sports and horseracing. A recent review of literature shows that there is yet a comprehensive screening method for antipsychoti...
Courtot D.At the request of the Service des Haras, our laboratory works on the toxicological problems of the sport-horse. These studies have resulted in the setting up of an anti-doping control for equestrian competitions of various types, not only flat racing. During events, horses, must be calm and docile to the riders' order. Frequently, the latter use tranquillizers to try and win events. The analytical method for the research and identification of these compounds is described. The technique involves successively: 1. alkalinisation of the sample - saliva, blood or urine after enzymatic hydrolysis. 2...
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for...
Karatt TK, Sathiq MA, Laya S, Philip M, Karakka Kal AK, Subhahar MB.An effective alternative to testosterone therapy is selective androgen receptor modulators, a class of compounds that has a tissue-specific effect on muscle and bone. These drugs, which enhance performance, pose a severe abuse risk in competitive sports. GLPG0492 is one of the selective androgen receptor modulators discovered in recent decades. This compound has a unique tissue-specific action for muscle and bone against steroid receptors and acts as a partial agonist for androgen receptors. This study examined GLPG0492 and its metabolites in vitro using equine liver microsomes. Liquid chromat...
Hertzsch R.Veterinarians play an essential role in the prevention of doping in equine sports, both as attending clinicians as well as official competition veterinarians. This complex task requires comprehensive knowledge concerning the pertinent regulations. These include official laws, such as the animal protection and medicines laws as as well as rules issued by the responsible equine sports authority. Furthermore, veterinarians are required to determine adequate withdrawal times following the legitimate use of medications in sport horses. This may be performed by applying appropriate data published by...
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.
Kwok WH, Choi TLS, Leung GNW, Wong ASY, Yue SK, Wan TSM, Ho ENM.The insulin-like peptide relaxin (RLX), an endogenous peptide hormone produced in human for pregnancy and reproduction, is also known to exert a range of physiological and pathological effects. Its use is banned in human sports, horseracing, and equestrian competitions due to its potential performance enhancing effect through vasodilation resulting in the increase of blood and oxygen supplies to muscles. Little is known about the biotransformation and elimination of RLX in horses. This paper describes an administration study of rhRLX-2 and its elimination in horses, and the development of sens...
Popot MA, Donval A, Bonnaire Y, Huau J.Feces are a possible medium to be used for horse doping control. Efficient methods for detecting drugs in feces collected from various animals are routinely applied in institutes of food safety in Belgium. We have already tested whether they are applicable to horse feces. In this report, accelerated solvent extraction (ASE), an efficient method for extracting compounds from solid material, has been tested. ASE has been used to replace the diethyl ether liquid-liquid extraction step present in the method initially set up. This technique has been optimized for detecting several non-steroidal ant...
Pinetre J, Delcourt V, Becher F, Garcia P, Barnabé A, Loup B, Popot MA, Fenaille F, Bailly-Chouriberry L.Many innovative biotherapeutics have been marketed in the last decade. Monoclonal antibodies (mAbs) and Fc-fusion proteins (Fc-proteins) have been developed for the treatment of diverse diseases (cancer, autoimmune diseases, and inflammatory disorders) and now represent an important part of targeted therapies. However, the ready availability of such biomolecules, sometimes characterized by their anabolic, anti-inflammatory, or erythropoiesis-stimulating properties, raises concerns about their potential misuse as performance enhancers for human and animal athletes. In equine doping control labo...
Haywood PE, Chalmers P.The chromatographic and spectroscopic properties of several unusual substances which have been detected in the "alkaloidal" chloroform extract from racehorse urine and saliva samples are reported. Some of these substances have been identified by combined gas chromatography-mass spectrometry and the source of the substance is stated where this is known. Other substances whose identity is not known have been detected and their mass spectra show characteristic amine fragments. The occurrence of these unidentified substances is more frequent in aged urine samples and it would therefore appear that...
Elbourne M, Cawley A, Stanley S, Bowen C, Fu S.Equine urine analysis has evolved over time to detect thousands of urinary compounds for doping control in the horse racing industry. The longitudinal assessment of 3-methoxytyramine to tyramine ratio (3-MT/T) values in equine urine by GC-MS profiling was investigated to support the Racing NSW Equine Biological Passport (EBP) for detection of dopaminergic manipulation in racehorses. This involved comparison of routine urine samples to administration studies of Sinemet, a common Parkinson's disease medication containing levodopa. Using an endogenous reference compound (ERC) in a urinary ratio e...
Harkins JD, Karpiesiuk W, Tobin T, Dirikolu L, Lehner AF.Ropivacaine is a local anesthetic that has a high potential for abuse in racing horses. It can be recovered from urine collected after administration as a hydroxylated metabolite following beta-glucuronidase treatment of the urine. Based on these findings, it has been inferred that ropivacaine is present in equine urine as a glucuronide metabolite; however, these metabolites have never been directly identified. Using ESI+/MS/MS, the presence of a [M+H]+ molecular ion of m/z 467 was demonstrated in urine corresponding to the calculated mass of a hydroxyropivacaine glucuronide +1. The abundance ...
McKinney AR.Control of the use of performance-affecting substances in the horse is critical to the integrity of a wide range of equine sports, with major implications for both animal welfare and revenue streams. One class of medications enjoying particular public notoriety is the anabolic-androgenic steroid group, as highlighted by the recent 'Big Brown' affair and Congressional inquiries into the use of steroids in professional sports, including horse racing, in the USA. This review examines the latest developments pertaining to the analytical detection of these substances in equine biological samples an...
Knych HK, Stanley SD, McKemie DS, Arthur RM, Bondesson U, Hedeland M, Thevis M, Kass PH.Although developed as a therapeutic medication, meldonium has found widespread use in human sports and was recently added to the World Anti-Doping Agency's list of prohibited substances. Its reported abuse potential in human sports has led to concern by regulatory authorities about the possible misuse of meldonium in equine athletics. The potential abuse in equine athletes along with the limited data available regarding the pharmacokinetics and pharmacodynamics of meldonium in horses necessitates further study. Eight exercised adult thoroughbred horses received a single oral dose of 3.5, 7.1, ...
