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.
Loup B, André F, Avignon J, Lhuaire M, Delcourt V, Barnabé A, Garcia P, Popot MA, Bailly-Chouriberry L.Short half-life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e., transcriptomics) has already shown interesting results in both humans and equines for erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroid (AAS) misuses. In humans, circulating cell-free microRNAs in plasma were described as new potential biomarkers for control of major doping agen...
Tozaki T, Ohnuma A, Iwai S, Kikuchi M, Ishige T, Kakoi H, Hirota K, Kusano K, Nagata S.Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene doping is to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness of digital PCR and real-time PCR in transgene detection using primers and probes that matched (P-true) or incompletely matched (P-false) the template DNA. Fluorescence intensity was significantly reduced when substituted probes were used compared to that using the matched probe in both digital and real-time P...
Caprioli G, Genangeli M, Mustafa AM, Petrelli R, Ricciutelli M, Sagratini G, Sartori S, Laus F, Vittori S, Cortese M.A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification ...
Witkowska-Piłaszewicz O, Pingwara R, Szczepaniak J, Winnicka A.Clenbuterol, the β2-adrenoceptor agonist, is gaining growing popularity because of its effects on weight loss (i.e., chemical liposuction). It is also popular in bodybuilding and professional sports, due to its effects that are similar to anabolic steroids. However, it is prohibited by anti-doping control. On the other hand, it is suggested that clenbuterol can inhibit the inflammatory process. The cells from 14 untrained and 14 well-trained race horses were collected after acute exercise and cultured with clenbuterol. The expressions of CD4, CD8, FoxP3, CD14, MHCII, and CD5 in PBMC, and reac...
Bishop R, Dzanis DA.This article provides reviews of the following: principal regulatory frameworks governing the supply of feed products for horses, focusing on the United States and Europe with guidance on compliance; key federal, state, or country requirements to ensure safe and accurately labeled products; rules concerning antidoping with a review of naturally occurring prohibited substances commonly found in feedstuffs; essential information for brand holders of equine nutrition products, practicing veterinarians, independent nutritionists, research scientists, competition riders and racehorse trainers, and ...
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...
Keen B, Cawley A, Fouracre C, Pyke J, Fu S.The emergence of novel doping agents is a continuous issue for analysts who aim to maintain the integrity of horseracing together with the well-being and safety of the animals and riders involved. Untargeted mass spectrometric analysis presents a potential improvement for antidoping as it enables the detection of compounds being indirectly affected by an administered drug. In this study, liquid chromatography-high-resolution mass spectrometry was used to investigate a 12-horse administration study of the synthetic opioid, butorphanol. A mass spectrometric workflow capable of detecting metaboli...
Mathew B, Philip M, Perwad Z, Karatt TK, Caveney MR, Subhahar MB, Karakka Kal AK.Hypoxia-inducible factor (HIF) stabilizer belongs to a novel class of pharmacologically active substances, which are capable of inducing the endogenous erythropoietic system. The transcriptional activator HIF has been shown to significantly increase blood hemoglobin and is well set for the treatment of anemia resulting from chronic kidney disease. This research work reports a comprehensive study of the most popular HIF stabilizer roxadustat and its metabolites in thoroughbred horse urine after oral administration. The plausible structures of the detected metabolites were postulated using liqui...
Trevisiol S, Moulard Y, Delcourt V, Jaubert M, Boyer S, Tendon S, Haryouli H, Taleb W, Caroff M, Chabot B, Drif L, André F, Garcia P, Loup B....According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organi...
Mikhail E, Siccardi E, Bawazir A, Rajesh A, Prathyush S, Al Wazani DMK, Sabeek M, John T.IOX-2 is a potent inhibitor of enzyme prolyl hydroxylases-2 (PHD) that plays a critical role in regulating hypoxia inducible factor (HIF) abundance and oxygen homeostasis. Federation for Equestrian Sports has listed HIF activators as prohibited substances to prevent their usage in doping. Consequently, it became essential to develop adequate knowledge and testing methods to detect it in equine sports drug testing samples. The validated method utilizes ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry in order to detect extremely low concentration of the ...
Broberg MN, Knych H, Bondesson U, Pettersson C, Stanley S, Thevis M, Hedeland M.Selective Androgen Receptor Modulators (SARMs) have anabolic properties but less adverse effects than anabolic androgenic steroids. They are prohibited in both equine and human sports and there have been several cases of SARMs findings reported over the last few years. The aim of this study was to investigate the metabolite profile of the SARM ACP-105 (2-chloro-4-[(3-endo)-3-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl]-3-methylbenzonitrile) in order to find analytical targets for doping control. Oral administration of ACP-105 was performed in horses, where blood and urine samples were collect...
Moreira F, Carmo H, Guedes de Pinho P, Bastos ML.Despite the impressive innate physical abilities of horses, camels, greyhounds, or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti-doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices pu...
Loganathan D, Yi R, Patel B, Zhang J, Kong N.Resolution of cathinone enantiomers in equine anti-doping analysis is becoming more important to distinguish the inadvertent ingestion of plant-based products from those of deliberate administration of designer synthetic analogs. With this in mind, a rapid and sensitive method was developed and validated for the detection, resolution and quantitative determination of cathinone enantiomers in horse blood plasma and urine. The analytes were recovered from the blood plasma and urine matrices by using a liquid-liquid extraction after adjusting the pH to 9. The recovered analytes were derivatized w...
Stucki M, Voegel CD, Binz TM, Kraemer T, Lavaud A, Voelter K.Equine sport agencies list steroids as prohibited substances for competing horses. Objective: The objective of this study was to investigate if the controlled substances dexamethasone and prednisolone are detectable in equine serum and urine samples during and after treatment with eye drops and if this can generate a positive doping test. Methods: Prospective cohort study. Methods: The study cohort included 11 horses. One eye of the horses was treated with either dexamethasone (Maxitrol 0.1%, n = 5 eyes) or prednisolone (Pred forte 1%, n = 6 eyes) eye drops 3 times daily for 14 days. Dexa...
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...
Bailly-Chouriberry L, Chu-Van E, Pinel G, Garcia P, Popot MA, André-Fontaine G, Bonnaire Y, Le Bizec B.Since the Australian commercialisation of the recombinant equine growth hormone (reGH) in 1998 (EquiGen-5), Bresagen), this reGH, which differs only from eGH by an additional methionine at the N-terminal end (met-eGH), is worldwide suspected to be administered to racehorses as a doping agent. Indeed, the use of this biological drug is considered as a threat to horseracing since it acts both on growth, development or reproductive functions, and on the improvement of performances. In this work, we describe two reliable techniques based on surface plasmon resonance biosensor immunoassay (SPR-BIA)...
Bailly-Chouriberry L, Baudoin F, Cormant F, Glavieux Y, Loup B, Garcia P, Popot MA, Bonnaire Y.The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal spe...
Thevis M, Opfermann G, Schänzer W.Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of eq...
Thevis M, Opfermann G, Krug O, Schänzer W.Isotope-dilution mass spectrometry has been employed successfully in numerous fields of analytical chemistry enabling the establishment of fast and reliable procedures. In equine sports, xanthine derivatives such as caffeine and theobromine are prohibited, and doping control laboratories analyze horse urine specimens regarding these illicit performance-enhancing drugs. Theobromine has to exceed a threshold level of 2 microg/mL, hence a robust and reliable quantitation is required. Stably deuterated theobromine and caffeine were synthesized by the reaction of xanthine or theobromine with iodome...
Bailly-Chouriberry L, Noguier F, Manchon L, Piquemal D, Garcia P, Popot MA, Bonnaire Y.Recombinant human erythropoietins (rHuEPOs) are glycoproteins drugs, produced by the pharmaceutical industry to restore production of red blood cells by stimulating human bone marrow for which this pathology has been diagnosed. It is suspected that these molecules are diverted as doping agents in horseracing to enhance oxygen transport and aerobic power in racehorses. Although indirect double-blotting or direct liquid chromatography-mass spectrometry (LC-MS) methods have been developed to confirm the presence of rHuEPO in a sample, the short detection time (48 h) is still a problem for doping ...
Kakoi H, Kijima-Suda I, Gawahara H, Kinoshita K, Tozaki T, Hirota K, Yoshizawa M.To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26-plex single-nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot(™) technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26-plex assay showed a probability of identity of 5.80 × 10(-11). Compared to the conventional short tan...
Loganathan D, Yi R, Patel B, Zhang J, Kong N.Resolution of cathinone enantiomers in equine anti-doping analysis is becoming more important to distinguish the inadvertent ingestion of plant-based products from those of deliberate administration of designer synthetic analogs. With this in mind, a rapid and sensitive method was developed and validated for the detection, resolution and quantitative determination of cathinone enantiomers in horse blood plasma and urine. The analytes were recovered from the blood plasma and urine matrices by using a liquid-liquid extraction after adjusting the pH to 9. The recovered analytes were derivatized w...
Guan F, You Y, Fay S, Li X, Robinson MA.To address the limitations of current targeted analytical methods that can only detect known doping agents, a novel methodology that permits untargeted drug detection (UDD) has been developed to help in the fight against doping in sports. Fifty-seven drugs were spiked into blank equine plasma and were treated as unknowns since their exact masses and chromatographic retention times were not utilized for detection. The spiked drugs were extracted from the plasma samples and were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). The acquired LC-HRMS raw ...
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...
Harding C, Viljanto M, Habershon-Butcher J, Taylor P, Scarth J.YK-11 is a steroidal selective androgen receptor modulator, a compound class prohibited in both equine racing and human sports because of their potentially performance enhancing properties. YK-11 is easily accessible via internet-based supplement vendors making this compound a possible candidate for doping; however, its phases I and II metabolism has not yet been reported in the horse. The purpose of this study was to investigate the in vivo metabolites of YK-11 in urine and plasma following oral administration with three daily doses of 50 mg to two Thoroughbred horses. In vitro incubations ...
Hansson A, Knych H, Stanley S, Thevis M, Bondesson U, Hedeland M.Selective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma. Methods: Each SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrome...
Lönnberg M, Bondesson U, Cormant F, Garcia P, Bonnaire Y, Carlsson J, Popot MA, Rollborn N, Råsbo K, Bailly-Chouriberry L.Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of e...
Ishii H, Shibuya M, So YM, Wong JKY, Ho ENM, Kusano K, Sone Y, Kamiya T, Wakuno A, Ito H, Miyata K, Yamada M, Leung GN.IOX4, a hypoxia-inducible factor stabilizer, is classified as a banned substance for horses in both horse racing and equestrian sports. We recently reported the pharmacokinetic profiles of IOX4 in horse plasma and urine and also identified potential monitoring targets for the doping control purpose. In this study, a long-term longitudinal analysis of IOX4 in horse hair after a nasoesophageal administration of IOX4 (500 mg/day for 3 days) to three thoroughbred mares is presented for the first time for controlling the abuse/misuse of IOX4. Six bunches of mane hair were collected at 0 (pre), ...
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...
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 ...
Abraham G, Allersmeier M, Gottschalk J, Schusser GF, Hoppen HO, Ungemach FR.There are no data available regarding the systemic (adverse) effects which might be induced by topical/dermal glucocorticoids (GCs) application in the horse. Besides their widespread use for the treatment of a variety of peripheral inflammatory disorders such as atopic dermatitis, eczemas or arthritis in the horse, their surreptitious application has become a concern in doping cases in competition/performance horses. Assessing both basal and ACTH-stimulated plasma cortisol as well as basal ACTH concentrations following application of dexamethsone-containing dermal ointment is necessary to dete...
Tozaki T, Ohnuma A, Nakamura K, Hano K, Takasu M, Takahashi Y, Tamura N, Sato F, Shimizu K, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA....The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/...
Delcourt V, Garcia P, Pottier I, Mansoibou N, Bache N, Glavieux Y, Chabot B, Perot I, André F, Loup B, Barnabé A, Popot MA, Bailly-Chouriberry L.Synthetic androgenic anabolic steroids (AAS) are banned compounds and considered as major threats by both racing and sports international authorities. Hence, doping control laboratories are continually looking into analytical improvements to increase their detection capabilities, notably by means of emerging technologies. To enhance analytical performances for the detection of synthetic AAS such as stanozolol, specific chromatographic procedures have been developed using recent quaternary liquid chromatography technology originally designed for high-throughput standardized proteomics connected...
Leung GN, Leung DK, Wan TS, Wong CH.This paper describes a high throughput LC-MS-MS method for the screening of 75 basic drugs in equine plasma at sub-ppb levels. The test scope covers diversified classes of drugs including some alpha- and beta-blockers, alpha- and beta-agonists, antihypotensives, antihypertensives, analgesics, antiarrhythmics, antidepressants, antidiabetics, antipsychotics, antiulcers, anxiolytics, bronchodilators, CNS stimulants, decongestants, sedatives, tranquilizers and vasodilators. A plasma sample was first deproteinated by addition of trichloroacetic acid. Basic drugs were then extracted by solid-phase e...
Ho EN, Kwok WH, Wong AS, Wan TS.A recent trend in the use of high resolution accurate mass screening (HRAMS) for doping control testing in both human and animal sports has emerged due to significant improvement in high resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution, and mass stability. A number of HRAMS methods have been reported for the detection of multi-drug residues in human or equine urine. As blood has become a common matrix for doping control analysis, especially in equine sports, a sensitive, fast and wide coverage screening method for detecting a large number of drugs in equine ...
Haughan J, Ortved KF, Robinson MA.Gene therapy uses genetic modification of cells to produce a therapeutic effect. Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression. Gene therapy is currently prohibited in bot...
Yuen BP, Wong KS, So YM, Kwok WH, Cheung HW, Wan TSM, Ho EN, Wong WT.Gene doping involves the misuse of genetic materials to alter an athlete's performance, which is banned at all times in both human and equine sports. Quantitative polymerase chain reaction (qPCR) assays have been used to control the misuse of transgenes in equine sports. Our laboratory recently developed and implemented duplex as well as multiplex qPCR assays for transgenes detection. To further advance gene doping control, we have developed for the first time a sensitive and definitive PCR-liquid chromatography high-resolution tandem mass spectrometry (PCR-LC-HRMS/MS) method for transgene det...
Ishii H, Shibuya M, Kusano K, Sone Y, Kamiya T, Wakuno A, Ito H, Miyata K, Yamada M, Leung GN.Daprodustat is a hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitor and is used as an erythropoiesis stimulant for the treatment of anemia in humans. In general, administering daprodustat to horses will result in a lifetime ban from both equestrian sports and horseracing by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. To control the misuse/abuse of daprodustat, we conducted nasoesophageal administration of daprodustat (100 mg/day for 3 days) to three thoroughbred mares and the post-administration hair ...
Garcia P, Paris AC, Gil J, Popot MA, Bonnaire Y.A sensitive method using LC/ESI-MS(n) has been developed on a quadrupole linear ion trap mass analyser for the detection of nine β(2) agonists (cimaterol, clenbuterol, fenoterol, formoterol, mabuterol, terbutaline, ractopamine, salbutamol and salmeterol) in horse urine. The method consists of solid-phase extraction on CSDAU cartridges before analysis by LC/ESI-MS(n) . The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allowed the detection of these compounds at pg/mL levels. Administration studies of fenoterol and formoterol are reported and show their pos...
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...
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...
Thevis M, Machnik M, Schenk I, Krug O, Piper T, Schänzer W, Düe M, Bondesson U, Hedeland M.The issue of illicit performance enhancement spans human and animal sport in presumably equal measure, with prohibited substances and methods of doping conveying both ways. Due to the proven capability of unbound ionic cobalt (Co(2) (+) ) to stimulate erythropoiesis in humans, both human and equine anti-doping regulations have listed cobalt as a banned substance, and in particular in horse drug testing, thresholds for cobalt concentrations applying to plasma and urine have been suggested or established. Recent reports about the finding of substantial amounts of undeclared nickel in arguably li...
Medana C, Calza P, Giancotti V, Dal Bello F, Pasello E, Montana M, Baiocchi C.Two horses were treated with sildenafil, and its metabolic products were sought in both urine and plasma samples. Prior to this, a simulative laboratory study had been done using a photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in the biological samples. The transformation of sildenafil and the formation of intermediate products were evaluated adopting titanium dioxide as photocatalyst. Several products were formed and characterized using the HPLC/HRMS(n) technique. The main intermediates identified in thes...
