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Topic:Doping
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.
Monitoring Estra-4,9-Diene-3,17-Dione Exposure in Equine Hair. This study investigated the incorporation of estra-4,9-diene-3,17-dione (dienedione) and its metabolites into equine hair following exogenous administration and evaluates the potential application of hair analysis for long-term doping surveillance. Mane hair samples were collected from three Thoroughbred castrated horses before and after oral administration of dienedione and from untreated colts for comparison. Dienedione was detected in post-administration hair from all treated horses but was absent in corresponding pre-administration samples. Segmental distribution patterns indicated contrib...
Detection and confirmation of hypoxia-inducible factor activating agents in equine plasma and urine by liquid chromatography coupled to mass spectrometry. Hypoxia-inducible factor (HIF) activating agents can promote the transcription of the erythropoietin gene, enhancing an athlete's performance, and are therefore prohibited in sports. A liquid chromatography coupled to high-resolution mass spectrometry (LCHRMS) method was developed for the detection and confirmation of 11 HIF-activating substances in equine plasma and urine. Ammonium formate added to the mobile phase system suppressed the responses of all the analytes in either positive or negative ion mode and thus was not used. The starting organic percentage in the mobile phase gradient and ...
Approaches to Thoroughbred Horse Identification in Diverse Sample Types Using Multiplexed SNP-PCR Sequencing Panels. The ability to determine the identity of equine samples would be highly advantageous in cases of ambiguous sample source, as individual-specific controls for gene editing detection, or forensic examination of suspect biological materials in potential doping cases. To this end, a SNP-PCR sequencing-based panel comprising 93 markers in one reaction was designed and investigated for use in Thoroughbred horses. Marker amplification performance and discriminating power were calculated against a population survey of 264 Thoroughbred whole blood samples from routine doping testing. The sensitivity of...
Detection of ionisable lipids in equine plasma by supported liquid extraction and liquid chromatography-high-resolution tandem mass spectrometry for doping control of mRNA agents. Messenger ribonucleic acid (mRNA) has emerged as a new class of therapeutic agent with unprecedented potential. In particular, the lipid nanoparticle (LNP)-mRNA formulations enabled safe and effective delivery of mRNA in vivo, leading to the success of mRNA vaccines during the COVID-19 pandemic. To control the potential misuse of LNP-mRNA agents in equine sports, a simple and sensitive method has been developed for the detection of ionisable lipids in equine plasma using supported liquid extraction (SLE) followed by liquid chromatography-high-resolution tandem mass spectrometry. To overcome th...
Administration Study of Bedinvetmab, an Antinerve Growth Factor Monoclonal Antibody, in Horses for Doping Control Purposes. Monoclonal antibodies (mAbs) targeting nerve growth factor (NGF) represent a novel therapeutic approach for managing acute and chronic pain in humans and animals. However, their potential misuse as pain-masking agents in competitive equine sports raises significant welfare and integrity concerns. To enhance understanding of the elimination profile of anti-NGF mAb if misused in horses, this study describes the first administration of bedinvetmab (a canine anti-NGF mAb) in three geldings, establishing its plasma elimination profile following subcutaneous administration. Bedinvetmab plasma concen...
Towards a Comprehensive Screening Approach for Detecting Gene Doping in Racehorses and Sport Horses by Targeting Common and Intrinsic Plasmid Sequences. The evolution of biotechnology and gene therapy has unfortunately facilitated the emergence of gene doping. Those misused applications are prohibited in equestrian sports (International Equestrian Federation (FEI)) and in horseracing (International Federation of Horseracing Authorities [IFHA]), reflecting major concerns regarding horse welfare and the integrity of competitions. Among the biotechnological tools that could be misused, eukaryotic expression plasmids represent a particular threat due to their ease of production, customisable design and ability to carry a wide range of performance ...
Plasma and urinary exposure of cannabidiol and cannabidiolic acid in horses following consumption of contaminated feed. Cannabidiol (CBD) and cannabidiolic acid (CBDA) are non-psychoactive cannabinoids naturally present in hemp-derived products. While these compounds are prohibited during equine competition, horses may be inadvertently exposed through contaminated feed. Data describing systemic and urinary exposure to CBD and CBDA following such low-dose, involuntary intake are limited. This study aimed to describe plasma and urine concentration-time profiles of CBD and CBDA in horses after consumption of experimentally contaminated feed. Results: Twelve Standardbred horses received feed containing low doses (1...
Investigation of Metformin Contamination in Horses: Implications for Environmental Exposure and Doping Control. Metformin is a diabetes medication with minimal therapeutic efficacy in veterinary medicine. However, recently there have been several adverse analytical findings in regulatory samples collected from racehorses. Metformin is commonly prescribed to humans, is excreted unchanged in urine, and is resistant to environmental degradation, raising concerns with respect to environmental contamination. In this study, six horses were exposed to shavings contaminated with metformin-positive urine at concentrations of 10, 135, and 270 μg/mL. Blood (plasma, serum, whole blood, and red blood cells) and u...
LC-HRMS detection of three ionizable lipids in equine plasma: surrogate markers for long-term LNP-mediated gene-doping surveillance. Lipid nanoparticles (LNPs) are essential delivery vehicles for messenger ribonucleic acid (mRNA)-based therapies. However, their potential misuse as gene-doping agents in horse racing and equestrian sports necessitates robust analytical surveillance. This study presents a highly sensitive, Orbitrap technology-driven liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the simultaneous detection of three clinically relevant ionizable lipids: ALC-0315, SM-102, and DLin-MC3-DMA (MC3), which are critical components of approved RNA therapeutics and vaccines. Following a modi...
Sensitive and selective quantification of elcatonin in equine plasma and urine using LC-FAIMS-MS/MS. The use of drugs for enhancing bone strength is prohibited under international regulations governing horse racing and equestrian sports. Elcatonin, a synthetic peptide with analgesic and bone-strengthening properties, is routinely monitored for compliance. However, its cyclic structure and high molecular weight hinder reliable detection. In this study, we developed and validated a sensitive liquid chromatography-high-field asymmetric waveform ion mobility spectrometry-tandem mass spectrometry method for quantifying elcatonin in equine plasma and urine. Sample preparation was optimised via weak...
Analytical Challenges and Emerging Detection Strategies of Synthetic Cannabinoids in Horse Doping Control. Synthetic cannabinoids (SCs) were originally synthesized to advance the understanding of the endocannabinoid system, facilitate disease research, and support the development of novel therapeutic agents. Compared with tetrahydrocannabinol, these compounds exhibit substantially higher psychoactive potency and enhanced receptor-binding affinity. The rapid and continuous evolution of SC derivatives presents significant challenges for analytical laboratories and increases the risk of misuse. Furthermore, SCs may be exploited to alter performance in both human and animal sports. The scarcity of comp...
Supramolecular Solvent Extraction for Doping Control Analysis of Prohibited Substances in Horse Urine. Despite the recent success in introducing supramolecular solvents (SUPRAS)-based extraction to drug analysis, its application and robustness in day-to-day regular urine testing have yet to be demonstrated. Moreover, the applicability of SUPRAS in equine doping control testing remains unexplored. In this work, we have successfully developed for the first time a simple, rapid, inexpensive, and environmentally friendly SUPRAS extraction method for analyzing 76 prohibited substances of different classes (selective androgen receptor modulators, hypoxia-inducible factor prolyl hydroxylase inhibitors...
Plasma and Urinary Elimination Profiles of Medetomidine Metabolites in Horses for the Purpose of Doping and Medication Control. Medetomidine, an α₂-adrenergic agonist, is widely used as a sedative in horses. While its pharmacological effects are established, limited data exist on elimination of its metabolites, 3'-hydroxy medetomidine (HMD) and 3'-carboxy medetomidine (CMD), which are of regulatory interest. HMD is currently targeted in plasma and urine under International Screening Limits (ISLs) established by the International Federation of Horseracing Authorities (IFHA). In this study, seven Thoroughbreds received 6.3 μg/kg of medetomidine intravenously. Blood and urine were collected for 96 h and analyzed b...
Metformin in the Horse: Pharmacokinetics and Detection Times Using Monte Carlo Simulations. The racehorse industry has strict regulations regarding the detection of prohibited substances in horses. Metformin, a diabetes medication, is a prohibited substance that has been reported in post-race blood and urine samples collected from racehorses. For further characterization of the disposition of metformin, 12 Thoroughbred horses were administered metformin orally and intravenously in a randomized, balanced, two-way crossover design. Serum and urine samples were collected, and drug concentrations determined via liquid chromatography-tandem mass spectrometry. The serum data were analyzed ...
Detection of Nandrolone Decanoate and Its Metabolites in Equine Hair After Intramuscular Administration. This paper describes the detection of nandrolone decanoate and its metabolites in mane hair collected from horses that have been treated with nandrolone decanoate (Deca-Durabolin). The intramuscular administration study of nandrolone decanoate in three Thoroughbred castrated horses (each received 800 mg weekly for 3 consecutive weeks) was previously conducted to investigate its metabolism and detection time in plasma and urine for doping control purposes. In this work, segmental analysis of the post-administration hair has revealed that (i) nandrolone decanoate and its metabolites, nandrolon...
Bioanalytical Uncertainty Assessment of Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry Method for Caffeine and Lidocaine in Equine Antidoping: A Dual Perspective on Bottom-up and Top-Down Approaches. The quality of quantitative results in bioanalysis requires not only a validated analytical method but also a rigorous estimation of measurement uncertainty. This study examines the challenges associated with the implementation of two distinct approaches in equine anti-doping control for the assessment of uncertainty associated with an ultra-high-performance liquid chromatography-high resolution mass spectrometry quantitative method for caffeine and lidocaine in horse urine. The bottom-up approach, based on the ISO Guide to the Expression of Uncertainty in Measurement (ISO GUM), was compared t...
Plasma and Urine Pharmacokinetics of Intravenous Pridinol in Thoroughbreds for Its Medication Control. We examined the pharmacokinetics of intravenous pridinol in six thoroughbred horses. Each horse received a single 20 mg dose of pridinol mesylate via the jugular vein, and plasma and urine samples were collected over 72 h. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify pridinol concentrations in plasma and urine, allowing for the calculation of pharmacokinetic parameters. A three-compartment model best fit the plasma elimination data. Using the Toutain model, irrelevant plasma and urine concentrations were estimated to be 0.00284 and 0.000612 ng/mL, respec...
Equine Metabolism of Voxelotor and Its Impact on Hematological Indices: A Doping Control Perspective. Voxelotor, a therapeutic drug for sickle cell disease, has been reported to elevate serum erythropoietin and hemoglobin levels in healthy individuals. Because of its potential to alter blood parameters, the World Anti-Doping Agency (WADA) classified voxelotor under category M1 of the 2023 Prohibited List. Despite this classification, little is known about its metabolic behavior in either humans or animals. In this study, the metabolism of voxelotor was investigated in Thoroughbred horses after oral administration. Using liquid chromatography high-resolution mass spectrometry (LC-HRMS), 35 meta...
Mapping the Metabolic Fate of Suzetrigine in Equine and Camel Models: A Step Toward Reliable Antidoping Detection. Effective pain management remains a persistent challenge, with opioids limited by tolerance, dependence, respiratory depression, and misuse. This has created demand for safer, nonaddictive alternatives. Suzetrigine (VX-548, Journavx) is a novel analgesic with promising efficacy, but its potential misuse in competitive racing requires investigation. Limited information is available on its metabolism and detection, emphasizing the need for metabolic characterization to aid antidoping strategies. Methods: In vitro metabolism of suzetrigine was evaluated using equine liver microsomes and homogeniz...
Development and validation of general urine screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high resolution mass spectrometry (LC-HRMS). The complexity of the drug market and the constant updating of drugs have been challenging issues for drug regulatory authorities. In this manuscript, a high-throughput automated assay based on Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 250-μL urine aliquot, which is subjected to enzymatic hydrolysis followed by Biotage Isolute supported liquid extraction plates using Biotage Extrahera system, evaporation, and reconstituti...
Suzetrigine in Equestrian Sports: Optimized Extraction and LC-HRMS Detection Strategies. Suzetrigine, a recently approved Na1.8 sodium channel blocker, shows strong potential in the treatment of neurological, psychiatric, and pain-related conditions. Its peripheral selectivity enables effective pain management while avoiding central nervous system complications and addiction risks linked to opioid use. Following FDA approval in January 2025, concerns have emerged regarding its possible misuse for performance enhancement in sports, highlighting the need for reliable detection tools in doping control. Methods: An analytical procedure was designed and validated to detect suzetrigine ...
Administration study of somapacitan, a long-acting growth hormone derivative, in horse for doping control purpose. Somapacitan is the second generation of recombinant human growth hormone (rhGH) medication that retains the pharmacological effects of rhGH but exhibits a longer duration of action due to its reversible albumin-binding in the body. In general, the use of all recombinant growth hormone (rGH) analogues is banned by the human and animal sports regulatory authorities due to their anabolic and lipolytic effects. However, little is known about the elimination kinetics and biological effects of the newly introduced long-acting rhGH, somapacitan, in horses. This paper describes the administration stud...
Metabolic Studies of Nandrolone Decanoate in Castrated Horses After Intramuscular Administration. This paper describes the studies of the in vitro biotransformation of nandrolone decanoate and its metabolic fate in equine plasma and urine after intramuscular administration to castrated thoroughbred horses. The in vitro metabolic study was performed using homogenised horse liver, and the more prominent in vitro biotransformation pathways were found to include hydrolysis, reduction, oxidation and sulfation, mainly resulting in seven Phase I metabolites and one Phase II metabolite. The administration study of nandrolone decanoate was carried out using three retired thoroughbred geldings, e...
Detection using chamber digital PCR with a DNA extraction-free method for gene-doping control. Gene doping, which entails the administration of transgenes, poses a serious threat to the integrity of equine sports and also raises both ethical and regulatory concerns. Current methods used for the detection of such doping often necessitate the extraction of DNA from plasma, which can be time-consuming and labour-intensive. To overcome this limitation, we developed a direct chamber digital PCR (cdPCR) method that enables transgene detection in equine plasma without the need for DNA purification. Using the equine erythropoietin (EPO) transgene as a model, we validated the assay by analysing ...
Analytical Data Review on an Artificial Intelligence Platform for Doping Control in Horse Racing. In the screening of prohibited substances (PS) in horse biological samples with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) for doping control, an enormous number of chromatograms are generated. Reviewing these chromatograms to identify suspicious findings requires an extensive manual effort. Recent advancements in Artificial Intelligence (AI) enable its use to classify images into different categories. This can potentially be utilized to perform first-line analysis of chromatograms, which are usually displayed as images, by classifying them...
Detection of Transdermal Application of Testosterone to Racehorses by Analysis of Urine and Plasma. The use of testosterone in racehorses is predominantly monitored using international urine and plasma concentration-based thresholds and complementary steroid ratios. To date, there has been no published pharmacokinetic study on transdermally applied testosterone products in horses and whether their use could result in adverse analytical findings. Therefore, quantitative analysis of testosterone and epitestosterone in urine and testosterone in plasma samples was performed following a pilot multi-dose transdermal Testogel administration (1 mg/kg once a day for 7 days on clipped skin) to one...
Development and validation of a quantitative UHPLC-HRMS bioanalytical method for equine anti-doping control. The quantification of banned substances in equine antidoping control, especially in racehorse urine, necessitates robust analytical methods with high detection levels due to the extremely low concentrations of the target substances and the significant impact of minor variations on doping test results. Reliable quantification is important for substances near regulatory thresholds, which, if exceeded, are prohibited. This study presents the development and validation of a bioanalytical UHPLC-HRMS method for quantifying doping substances in equine urine, including diazepam and acepromazine with a...
Doping Control of Ranitidine in Horses. Ranitidine is a histamine H-receptor antagonist commonly used to treat gastric ulceration in horses. The author's laboratory conducted a study some years ago in the early 2000s on its metabolism as well as its urinary elimination profile in two geldings. With the technology advancement as well as popularity of blood for doping control testing, the laboratory has recently conducted another administration trials of the substance in six horses to study the in vivo metabolism of ranitidine, aiming to identify and reinvestigate the appropriate target(s) for controlling misuse of ranitidine in hors...
Multiplex PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry for Simultaneous Detection of Multiple Transgenes in Equine Plasma. The development of gene therapy techniques introduces a potential risk of gene doping, which threatens the integrity of sport. In response to this challenge, we have developed a novel analytical method that employs a multiplex polymerase chain reaction (PCR) in conjunction with liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) for the simultaneous identification of multiple transgenes in equine plasma within a single reaction. The method targets three potential doping transgenes: equine growth hormone 1 (eGH1), equine growth hormone-releasing hormone (eGHRH), and equi...
Pooled Sampling Technique to Improve the Monitoring of Medication Use in the Racehorse Industry. All anti-doping programmes face financial constraints and monitoring trends in medication use or abuse in a population of racehorses can be difficult and expensive. Obtaining biological samples is the primary method of anti-doping control in individual horses or stables of horses but can be invasive and expensive. Another important practice of anti-doping control has been the confiscation of used and filled syringes by regulators for individual forensic analysis. Pooled samples testing involves the testing of multiple individual samples together as one composite sample. This pooled sample appr...
