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.
Barnabé A, Moulard Y, Trevisiol S, Boyer S, Caroff M, Taleb W, Tendon S, Drif L, Delcourt V, Popot MA, Bailly-Chouriberry L.The study focuses on the discovery of kavain, a natural-occurring compound of the plant Piper methysticum, in a post-race urine sample of a horse. The researchers traced the potential source […]
Ishii H, Shibuya M, Leung GN, Nozawa S, Yamashita S, Yamada M, Kushiro A, Kasashima Y, Okada J, Kawasaki K, Kijima-Suda I.GW1516 is a peroxisome proliferator-activated receptor-δ (PPAR-δ) agonist that is banned in horseracing and equestrian sports. Long-term detection and longitudinal distribution of GW1516 in the mane of a horse are reported for the first time and this hair analysis could prolong the detection window of GW1516 for doping control. Methods: Mane hairs were divided into three segments (0-7, 7-15, and >15 cm from the cut end) and completely pulverized and homogenized for analysis. The pulverized hair samples were extracted with methanol followed by further purification and the extracts were a...
Cheung HW, Wong KS, To NS, Bond AJ, Farrington AF, Prabhu A, Curl P, Wan TSM, Ho ENM.Selective androgen receptor (AR) modulators (SARMs) are potent anabolic agents with a high potential of misuse in horseracing and equestrian sports. In this study, we applied label-free proteomics to discover plasma protein biomarkers in geldings (castrated horses) after administration with a popular SARM named RAD140. Tryptic peptides were prepared from plasma samples and analyzed by nano-flow ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (nano-UHPLC-HRMS/MS) using data-independent acquisition (DIA) method. Orthogonal projection on latent structure-discr...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Kusano K, Nagata SI.Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene and genome editing detection methods based on whole genome resequencing (WGR) using genomic DNA extracted from whole blood. In this study, we aimed to develop a WGR method using DNA extracts from hair roots. Hair roots are a preferred substrate because their collection is less invasive than blood collection. Hair is also easier to store for long periods of time. Although almost all genomic DNA extracted from hair root samples stored for years at room temperature was degraded...
Ishii H, Shibuya M, Leung GN, Yamashita S, Yamada M, Kushiro A, Kasashima Y, Okada J, Kawasaki K, Kijima-Suda I.The use of GW1516, a peroxisome proliferator-activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine. Methods: Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein-precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) with a Q-E...
Johnson E, van Heemst J, Benavides J, Gray B.Etizolam is a benzodiazepine analogue that is approved for use in Japan, Italy and India but has recently appeared as a nonapproved product on the illicit drug market in Europe and North America. Etizolam was identified in a crystalline material seized at a Kentucky racetrack, raising concerns that this drug may have been used in racing. The aim of this study was to characterize the metabolism and excretion of etizolam in horses to generate information on its disposition and to incorporate the correct urinary and serum target analytes into anti-doping screening procedures. Etizolam was adminis...
Moeller BC, Yang Z.Controlling the abuse of prohibited substances such as anabolic steroids, selective androgen receptor modulators, β-adrenoceptor agonists, and blood doping agents is of great interest to racing authorities. The use of dried blood spots (DBS) as an alternative sampling approach may be a feasible approach for controlling the use of these agents. To assess the feasibility of using DBS in equine blood, an 11-min liquid chromatography-mass spectrometry method was developed on a triple quadrupole mass spectrometer following extraction from Whatman 903 DBS cards. A total of 50 compounds across multi...
Cutler C, Viljanto M, Taylor P, Habershon-Butcher J, Muir T, Biddle S, Van Eenoo P.AC-262536 is one of a number of selective androgen receptor modulators that are being developed by the pharmaceutical industry for treatment of a range of clinical conditions including androgen replacement therapy. Though not available therapeutically, selective androgen receptor modulators are widely available to purchase online as (illegal) supplement products. The growth- and bone-promoting effects, along with fewer associated negative side effects compared with anabolic-androgenic steroids, make these compounds a significant threat with regard to doping control in sport. The aim of this st...
Cheung HW, Wong KS, Lin VYC, Wan TSM, Ho ENM.The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, ...
Subhahar MB, Karakka Kal AK, Philip M, K Karatt T, N I, Vazhat RA, M P MA.ACP-105 is a novel nonsteroidal selective androgen receptor modulator (SARM) with a tissue-specific agonist effect and does not have side effects associated with the use of common androgens. This research reports a comprehensive study for the detection of ACP-105 and its metabolites in racehorses after oral administration (in vivo) and postulating its structures using mass spectrometric techniques. To obtain the metabolic profile of ACP-105, a selective and reliable LC-MS/MS method was developed. The chemical structures of the metabolites were determined based on their fragmentation pattern, a...
Ishii H, Leung GN, Yamashita S, Yamada M, Kushiro A, Kasashima Y, Okada J, Kawasaki K, Kijima-Suda I.GW1516 is a peroxisome proliferator-activated receptor-δ agonist in the class of hormones and metabolic modulators. The use of GW1516 is banned in both horseracing and equestrian competitions. To the best of our knowledge, this is the first metabolic study of GW1516 in horses. Methods: After protein precipitation of pre- and post-administration plasma GW1516 samples, the supernatants were analyzed using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry to detect GW1516 and its metabolites. Monoisotopic ions of GW1516 and its metabolites were monitored ...
Tozaki T, Ohnuma A, Takasu M, Nakamura K, Kikuchi M, Ishige T, Kakoi H, Hirora KI, Tamura N, Kusano K, Nagata SI.Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetica...
Chan GHM, Tang W, Curl P, Lin Y, Wan TSM, Ho ENM.Arsenic can be easily found in our surrounding environment. Because of its ubiquitous nature, horse urine and blood invariably contain low levels of arsenic. Nevertheless, inorganic arsenic, despite its general use as a tonic for horses, is an effective doping agent having a deleterious effect because of its ability to induce gastroenteritis. The misuse of arsenic in horseracing has been controlled by an international urinary threshold of total arsenic at 0.3 μg/mL. However, an equivalent threshold for total arsenic in plasma is yet to be established. In this study, an inductively coupled pla...
Kwok KY, Choi TLS, Kwok WH, Lau MY, Leung EMK, Leung GNW, Wong JKY, Wan TSM, Adrian FF, Prabhu A, Ho ENM.The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non-peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was...
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 ...
Garcia P, Pinètre J, Morel S, Jaubert M, Deruy X, Perot I, Delcourt V, Loup B, Popot MA, Bailly-Chouriberry L.Bisphosphonates are prohibited drugs according to Article 6 of the International Agreement on Breeding, Racing and Wagering of the International Federation of Horseracing Authorities (IFHA) and the International Equestrian Federation (FEI). These compounds are used for the treatment of lameness, navicular and bone diseases in horses and are divided into two groups: non-nitrogen-containing bisphosphonate drugs (e.g. clodronic acid) and nitrogen-containing bisphosphonate drugs (e.g. zoledronic acid). Their hydrophilic properties and the high affinity for the bone matrix make the control of their...
Göktaş EF, Kabil E, Arıöz F.Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in therapeutic doses in human and veterinary medicine for the treatment of inflammation, pain, and fever. A method for the simultaneous determination of nine NSAIDs, known as therapeutic prohibited substances, in equine urine was developed and fully validated according to the European Commission Decision 2002/657/EC and Association of Official Racing Chemists criteria. The validation was performed for naproxen, flunixin, ketoprofen, diclofenac, eltenac, meclofenamic acid, phenylbutazone, vedaprofen, and carprofen in equine urine i...
Ishii H, Obara T, Kusano K, Kijima-Suda I.Paracetamol is commonly used as an over-the-counter analgesic and antipyretic medication for humans, but not sold as a legitimate therapeutic medication for horses in Japan. However, paracetamol is commonly found in horses together with its two isomers, metacetamol and orthocetamol. We previously reported that paracetamol and orthocetamol were both present in selected feed consumed by Japanese racehorses. For the purpose of the doping control of paracetamol in local Japanese horses, we proposed establishing residue limits (Japanese residue limits, JRLs) to minimize the risk of reporting parace...
Waraksa E, Owczarek K, Kubica P, Kłodzińska E, Ozimek M, Wrzesień R, Bobrowska-Korczak B, Namieśnik J.Bromhexine (BH), expectorant used in the treatment of respiratory disorders associated with viscid or excessive mucus, is not permitted for use in the competing horse by many authorities in horseracing and Olympic disciplines. Metabolic studies are of the great importance in anti-doping field because they allow for updating the selection of the most appropriate markers for prohibited substances, such as metabolites present at higher concentration levels and/or lasted for a longer period of time in biological samples than a parent drug. This study describes LC-MS/MS-based method for simultaneou...
Gadaj A, Ventura E, Healy J, Botrè F, Sterk SS, Buckley T, Mooney MH.Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (β-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacop...
Wong JKY, Chan GHM, Choi TLS, Kwok KY, Lau MY, Leung GNW, Wan TSM, Ho ENM.A high-throughput method has been developed for the doping control analysis of 124 drug targets, processing up to 154 horse urine samples in as short as 4.5 h, from the time the samples arrive at the laboratory to the reporting deadline of 30 min before the first race, including sample receipt and registration, preparation and instrument analysis and data vetting time. Sample preparation involves a brief enzyme hydrolysis step (30 min) to detect both free and glucuronide-conjugated drug targets. This is followed by extraction using solid-supported liquid extraction (SLE) and analysis using liq...
Mizobe F, Mori M, Nagata SI, Yamashita S, Okada J, Kusano K.Ever since 'One Health' concept was introduced in early 2000s, judicious use of antimicrobials by veterinarians has become an issue of great concern. Recently, findings of anti-inflammatory effects in certain types of antimicrobials have raised a subject for discussion among racing authorities. Regulatory framework of antimicrobials in racing should be based on best interest of horse welfare and doping control perspective, but basic data on prevalence of antimicrobials are lacking. Analysis of 100 postrace urinary samples collected from 10 Japanese racecourses by targeting 21 antimicrobials us...
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...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan prob...
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...
Guan F, Fay S, Li X, You Y, Robinson MA.Bioactive peptides pose a great threat to sports integrity. The detection of these peptides is essential for enforcing their prohibition in sports. Identifying the catabolites of these peptides that are formed ex vivo in plasma may improve their detection. In the present study, the stability of 27 bioactive peptides with protection at both termini in equine plasma was examined under different incubation conditions, using HILIC coupled to HRMS. Of the 27 peptides, 13 were stable after incubation at 37°C for 72 hr, but the remaining 14 were less stable. Ex vivo catabolites of these 14 peptides ...
Haughan J, Jiang Z, Stefanovski D, Moss KL, Ortved KF, Robinson MA.Gene therapy promotes the expression of missing or defective genes and can be curative following administration of a single dose. Gene therapy is prohibited in equine athletes by regulatory bodies due to the high potential for abuse and novel analytical methods are needed for detection. The goal of this study was to detect the administration of an experimental gene therapy: a recombinant adeno-associated viral vector (rAAV) carrying a transgene for the anti-inflammatory cytokine IL-10 (rAAV-IL10). Twelve horses were randomly assigned to receive an intra-articular injection of rAAV-IL10 or phos...
Wong KS, Cheung HW, Choi TLS, Kwok WH, Curl P, Mechie SC, Prabhu A, Wan TSM, Ho ENM.Recent advances in label-free quantitative proteomics may support its application in identifying and monitoring biomarkers for the purpose of doping control in equine sports. In this study, we developed a workflow of label-free quantitative proteomics to propose plasma protein biomarkers in horses after administration with krypton (Kr), a potential erythropoiesis-stimulating agent. Plasma proteomes were profiled by using nanoliquid chromatography-high-resolution mass spectrometry. An in-house mass spectral library consisting of 1121 proteins was compiled using samples collected from geldings (...
Karakka Kal AK, Perwad Z, K Karatt T, Nalakath J, Subhahar M.Recently, an increased tendency to use various metals has been observed in the sports competition fields. Many of these metals and their organic complexes reportedly have good pharmacologic, therapeutic and performance-enhancement uses; they are banned or recommended as controlled medications in competitive sports. The objective of this research was to determine the concentration of pharmacologically relevant metals in urine samples collected from racehorses at various sport events, develop a method and assess the concentrations of above metals using inductively coupled plasma mass spectrometr...
Waller CC, Weththasinghe SA, McClure L, Cawley AT, Suann C, Suann E, Sutherland E, Cooper E, Heather A, McLeod MD.Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17β-ol), reduced and dihydroxylated madol (17α-me...
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, 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).
Ishii H, Shibuya M, Kusano K, Sone Y, Kamiya T, Wakuno A, Ito H, Miyata K, Sato F, Kuroda T, Yamada M, Leung GN.In drug metabolism studies in horses, non-targeted analysis by means of liquid chromatography coupled with high-resolution mass spectrometry with data-dependent acquisition (DDA) has recently become increasingly popular for rapid identification of potential biomarkers in post-administration biological samples. However, the most commonly encountered problem is the presence of highly abundant interfering components that co-elute with the target substances, especially if the concentrations of these substances are relatively low. In this study, we evaluated the possibility of expanding DDA coverag...
Hansson A, Knych H, Stanley S, Thevis M, Bondesson U, Hedeland M.Selective androgen receptor modulators, SARMs, constitute a class of compounds with anabolic properties but with few androgenic side-effects. This makes them possible substances of abuse and the World Anti-Doping Agency (WADA) has banned the entire class of substances. There have been several cases of illicit use of aryl propionamide SARMs in human sports and in 2013, 13 cases were reported. These substances have been found to be extensively metabolized in humans, making detection of metabolites necessary for doping control. SARMs are also of great interest to equine doping control, but the in...
Liu Y, Uboh CE, Li X, Guan F, You Y, Maylin GA, Zhu F, Soma LR.A rapid and sensitive method for simultaneous screening, quantification and confirmation of 17 barbiturates in horse plasma using liquid chromatography-tandem mass spectrometry is described. Analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether, separated on a C18 column, and analyzed in negative electrospray ionization mode. Multiple-reaction monitoring was employed for screening and quantification. Confirmation for the presence of the analytes was achieved by comparing ion intensity ratio. The ranges for limits of detection, quantification and confirma...
Johansson M, Anlér EL.A quantitative method for the analysis of flunixin, 2-(2-methyl-3-trifluoromethylanilino) nicotinic acid, in equine urine by gas chromatography with nitrogen-phosphorus detection has been developed. Flunixin and the internal standard, mefenamic acid, N-(2,3-xylyl) anthranilic acid, were analysed after extractive methylation of the carboxylic acid group using methyl iodide. The extraction and alkylation conditions of flunixin and mefenamic acid have been studied. The detection limit of the method was 0.25 mumol/l flunixin in urine (74 ng/ml). Flunixin was found to be conjugated to 96.5% in equi...
Popot MA, Ho ENM, Stojiljkovic N, Bagilet F, Remy P, Maciejewski P, Loup B, Chan GHM, Hargrave S, Arthur RM, Russo C, White J, Hincks P, Pearce C....Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma stud...
Yu NH, Ho EN, Tang FP, Wan TS, Wong AS.A multi-target high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of low ppt to low ppb levels of anabolic steroids, corticosteroids, anti-diabetics, and non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma was developed for the purpose of doping control. Plasma samples were first deproteinated by addition of trichloroacetic acid. Drugs were then extracted by solid-phase extraction (SPE) using Bond Elut Certify cartridges, and the extracts were analysed by a triple-quadrupole/linear ion trap LC-MS-MS instrument in positive electrospray...
Eenoo PV, Delbeke FT.Salbutamol is a beta-adrenergic agonist that is used in the treatment of asthma in humans and chronic obstructive pulmonary disease in horses. Because of its stimulating and growth promoting properties, it is prohibited by horse racing authorities. Recently a number of adapters (eg Equinehaler) have been designed, allowing the use of metered dose inhalers (MDI) approved for human use. However, information on detection times of salbutamol after administration of salbutamol in therapeutic doses by inhalation is lacking. In this study, 2 mg salbutamol (Ventolin) was administered to four standardb...
Yu NH, Ho EN, Leung DK, Wan TS.Anabolic steroids have the capability of improving athletic performance and are banned substances in the Olympic games as well as in horseracing and equestrian competitions. The control of their abuse in racehorses is traditionally performed by detecting the presence of anabolic steroids and/or their metabolite(s) in urine samples using gas chromatography-mass spectrometry (GC-MS). However, this approach usually requires tedious sample processing and chemical derivatisation steps and could be very insensitive in detecting certain steroids. This paper describes a high performance liquid chromat...
Wieder ME, Paine SW, Hincks PR, Pearce CM, Scarth J, Hillyer L.Salbutamol sulphate (Ventolin Evohaler) was administrated via the inhalation route to six horses at a dose of 0.5 mg every 4 h during the day for 2 days (total dose 4 mg). Urine and blood samples were taken up to 92 h postadministration. Hydrolyzed plasma and urine were extracted using solid phase extraction (SPE). A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification (LLOQ) for salbutamol of 10 pg/mL in plasma and urine. The parent drug was identified using UPLC-MS/MS. Most of the determined salbutamol plasma concentrations, post l...
Clarke A, Scarth J, Teale P, Pearce C, Hillyer L.Detection of androgenic-anabolic steroid abuse in equine sports requires knowledge of the drug's metabolism in order to target appropriate metabolites, especially where urine is the matrix of choice. Studying 'designer' steroid metabolism is problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of toxicological data. In this study, the equine in vitro metabolism of eight steroids available for purchase on the Internet is reported; including androsta-1,4,6-triene-3,17-dione, 4-chloro,17α-methyl-androsta-1,4-diene-3,17β-diol, estra-4,9-diene-...
Current drug metabolismAugust 27, 2022
Volume 23, Issue 10 850-865 doi: 10.2174/1389200223666220825093945
Ishii H, Shibuya M, Kusano K, Sone Y, Kamiya T, Wakuno A, Ito H, Miyata K, Sato F, Kuroda T, Yamada M, Leung GN.Vadadustat, a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, is a substance which carries a lifetime ban in both horse racing and equestrian competition. A comprehensive metabolic study of vadadustat in horses has not been previously reported. Objective: Metabolism and elimination profiles of vadadustat in equine plasma and urine were studied for the purpose of doping control. Methods: A nasoesophageal administration of vadadustat (3 g/day for 3 days) was conducted on three thoroughbred mares. Potential metabolites were comprehensively detected by differential analysis of ful...
Kwok WH, Choi TL, So PK, Yao ZP, Wan TS.Xenon can activate the hypoxia-inducible factors (HIFs). As such, it has been allegedly used in human sports for increasing erythropoiesis. Krypton, another noble gas with reported narcosis effect, can also be expected to be a potential and less expensive erythropoiesis stimulating agent. This has raised concern about the misuse of noble gases as doping agents in equine sports. The aim of the present study is to establish a method for the simultaneous detection of xenon and krypton in equine plasma for the purpose of doping control. Xenon- or krypton-fortified equine plasma samples were prepar...
Maniego J, Pesko B, Habershon-Butcher J, Hincks P, Taylor P, Tozaki T, Ohnuma A, Stewart G, Proudman C, Ryder E.Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction in domestic animals but also in livestock production by enhancement of desirable traits. The existence of the technology, however, leaves it open to potential misuse in performance-led sports such as horseracing and other equestrian events. Recent advances in equine gene editing, regarding the generation of gene-edited embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer, have highlighted the need to develop tools to detect potential prohibited use of the technology. On...
Habershon-Butcher J, Cutler C, Viljanto M, Hincks PR, Biddle S, Paine SW.Xylazine is widely used worldwide as a short-acting sedative in general equine and racing practice. In the UK, although it has a legitimate use during training, equine anti-doping rules state it is a prohibited substance on race day. The aim of the study was to produce a detection time (DT) to better inform European veterinary surgeons so that xylazine can be used appropriately under regulatory rules. Previous publications have various limitations pertaining to analysis method, particularly for plasma and limited length of time of sample collection. In this study, pharmacokinetic data were pro...
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...
Garcia P, Pinètre J, Morel S, Jaubert M, Deruy X, Perot I, Delcourt V, Loup B, Popot MA, Bailly-Chouriberry L.Bisphosphonates are prohibited drugs according to Article 6 of the International Agreement on Breeding, Racing and Wagering of the International Federation of Horseracing Authorities (IFHA) and the International Equestrian Federation (FEI). These compounds are used for the treatment of lameness, navicular and bone diseases in horses and are divided into two groups: non-nitrogen-containing bisphosphonate drugs (e.g. clodronic acid) and nitrogen-containing bisphosphonate drugs (e.g. zoledronic acid). Their hydrophilic properties and the high affinity for the bone matrix make the control of their...
Guan F, You Y, Li X, Robinson MA.Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty-four peptides were extracted using mixed-mode solid-phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state ...
Yamada M, Aramaki S, Kurosawa M, Kijima-Suda I, Saito K, Nakazawa H.The use of anabolic steroids in racehorses is strictly regulated. We have developed a method for the simultaneous analysis of 11 anabolic steroids: fluoxymesterone, 17alpha-methyltestosterone, mestanolone, methandienone, methandriol, oxymetholone, boldenone, furazabol, methenolone, nandrolone, and stanozolol, for possible application to a doping test in racehorses. We selected 15 kinds of target substances for a doping test from the main metabolites of these anabolic steroids, and established a method for simultaneous analysis. Urine was hydrolyzed and subjected to solid-phase extraction. Then...
Taddei L, Benoit M, Sukta A, Peterson J, Gaensslen RE, Negrusz A.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 ...
Stanley SM, Poljak A.Mass spectrometric analyses of the recombinant proteins in Eprex and Aranesp were undertaken with the goal of producing reference mass spectra and evaluating strategies to improve its applicability as a method for equine and canine doping control of these substances. A simple, low chemical noise deglycosylation reaction removed microheterogeneity due to post-translational carbohydrate attachment and both proteins were detectable using MALDI-TOF-MS. Deglycosylated human erythropoietin (hEPO) was also detected using HPLC-ESI-MS. This is the first time that spectra of deglycosylated Eprex and Ara...
Lotfollahzadeh S, Mokhber-Dezfouli MR, Tajik P, Bokaie S, Watson DG.The present survey evaluated the use of prohibited substances cases in the first 2 years of medication regulation in horseracing in Iran so that the impact of these regulations on the level of positive cases over the period could be assessed. Objective: To determine the prevalence of positive tests for prohibited substances in horse races during 2 years of a drugs testing programme in Iran. Methods: A total of 656 horses that were winners or second in races were tested during the 2 year study. In the first year 354 horses (209 males and 145 females) and in the second year 302 horses (155 males...
Genangeli M, Caprioli G, Cortese M, Laus F, Petrelli R, Ricciutelli M, Sagratini G, Sartori S, Vittori S.A new fast and easy analytical procedure for the simultaneous detection and quantification of 9 anabolic steroids (deslorelin, dexamethasone sodium phosphate, prednisolone, methylprednisolone, stanozolol, boldenone, nandrolone, dexamethasone isonicotinate and altrenogest) in horse urine for doping control have been developed by using the ultra-high-performance liquid chromatography coupled with tandem mass spectrometry technique (UHPLC-MS/MS). A total amount of 400 μl of sample was evaporated, restored and injected in the UHPLC-MS/MS. The proposed method was fully validated showing a recove...
Piper T, Geyer H, Gougoulidis V, Flenker U, Schänzer W.Boldenone (androsta-1,4-dien-17beta-ol-3-one, Bo) is an anabolic steroid known to have been used in cattle breeding or equine sport as a doping agent for many years. Although not clinically approved for human application, Bo or its main metabolite 5beta-androst-1-en-17beta-ol-3-one (BM1) were detected in several doping control samples. For more than 15 years the possibility of endogenous Bo production in human beings has been discussed. This is a challenging issue for doping control laboratories as Bo belongs to the list of prohibited substances of the World Anti-Doping Agency and therefore th...
Hertzsch R, Richter A.Based on their performance-enhancing potential, caffeine and theophylline are prohibited substances in equine sports. Residues in horses can be caused by wilful application or by unintended uptake of contaminated feed. The International Federation of Horseracing Authorities recently introduced international residue limits (IRLs) to facilitate the discrimination between pharmacological relevant and irrelevant concentrations in doping samples. The objective of this study was to investigate the scientific validity of these IRLs. A systematic analysis was performed to assess the IRLs by different ...
Stojiljkovic N, Leroux F, Bubanj S, Popot MA, Paris A, Tabet JC, Junot C.There is an urgent need to implement holistic and untargeted doping control protocols with improved discriminatory power, compared to conventional methods that only target doping agents. Metabolomics, which aims to characterize all metabolites present in biological matrices, could fulfill this need. In this context, the aim of this study was to evaluate the impact of environmental factors on the ability to obtain a metabolic signature of stanozolol administration in horse doping situation. Urine samples from 16 horses breeded in two different places were collected over a one-year period, befor...
You Y, Proctor RM, Guo K, Li X, Xue E, Guan F, Robinson MA.High-resolution mass spectrometry (HRMS) is a very powerful technology for equine doping control analysis. The more recently developed hybrid type of Orbitrap-based HRMS instrument allows for both targeted and non-targeted screening analyses in a single liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) run. In the present study, an LC-HRMS/MS method was developed and validated to detect prohibited substances in equine sports. The substances were recovered from equine plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether and were separated on a C18 rev...
Jaussaud P, Courtot D, Guyot JL, Paris J.The main metabolite of flunixin, a hydroxylated product, has been identified by gas chromatography-mass spectrometry and 1H NMR spectroscopy in equine urine and plasma. The method also permits the qualitative monitoring of the urinary elimination of the drug and its metabolite. The two products are detected up to 175 and 54 h, respectively, after a single intravenous administration at the dose of 1 mg/kg. Simultaneous detection of the two compounds increases the reliability of anti-doping control analysis.
Cloteau C, Dervilly G, Loup B, Delcourt V, Kaabia Z, Bagilet F, Groseille G, Dauriac K, Fisher S, Popot MA, Garcia P, Le Bizec B, Bailly-Chouriberry L.Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work focuses on assessing the robustness of the associated method and def...
