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Sha Tin, China

Wong J.K.Y.,Racing Laboratory | Wan T.S.M.,Racing Laboratory
Veterinary Journal | Year: 2014

Doping. Doping can be either intentional or unintentional, which will be the meaning adopted in this review. This is in line with the World Anti-Doping Agency's definition of doping, namely, a violation of the rule(s) controlling the use of prohibited substances or practices.1 in sports is highly detrimental, not only to the athletes involved but to the sport itself as well as to the confidence of the spectators and other participants. To protect the integrity of any sport, there must be in place an effective doping control program. In human sports, a 'top-down' and generally unified approach is taken where the rules and regulations against doping for the majority of elite sport events held in any country are governed by the World Anti-Doping Agency (WADA). However, in horseracing, there is no single organisation regulating this form of equestrian sport; instead, the rules and regulations are provided by individual racing authorities and so huge variations exist in the doping control programs currently in force around the world.This review summarises the current status of doping control analyses in horseracing, from sample collection, to the analyses of the samples, and to the need for harmonisation as well as exploring some of the difficulties currently faced by racing authorities, racing chemists and regulatory veterinarians worldwide. © 2014 Elsevier Ltd. Source

Ho E.N.M.,Racing Laboratory | Wan T.S.M.,Racing Laboratory | Wong A.S.Y.,Racing Laboratory | Lam K.K.H.,Veterinary Regulation and International Liaison | Stewart B.D.,Veterinary Regulation and International Liaison
Journal of Chromatography A | Year: 2011

Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05. ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4. h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples. © 2010 Elsevier B.V. Source

Wong A.S.Y.,Racing Laboratory | Ho E.N.M.,Racing Laboratory | Wan T.S.M.,Racing Laboratory
Drug Testing and Analysis | Year: 2012

Myo-inositol trispyrophosphate (ITPP) is a new drug capable of increasing the amount of oxygen in hypoxic tissues. Studies have shown that administration of ITPP increases the maximal exercise capacity in normal mice as well as mice with severe heart failure. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. While there have been speculations in the horseracing industry that the covert use of ITPP is already widespread, no reported method exists for the detection of ITPP in equine biological samples. ITPP is a difficult-to-detect drug due to its hydrophilic nature; the complexity of equine biological matrices also adds to the problem. This paper describes for the first time a method for the detection and confirmation of ITPP in equine urine and plasma. ITPP was isolated from the sample matrices by solid-phase extraction and the extract was analyzed by hydrophilic interaction chromatography-tandem mass spectrometry. ITPP could be detected at low ppb levels in both fortified equine plasma and urine with good precision, fast instrumental turnaround time, and negligible matrix interferences. To our knowledge, this is the first report of a validated method for the detection and unequivocal confirmation of low levels of ITPP in any biological fluid. © 2012 John Wiley & Sons, Ltd. Source

Wong C.H.F.,Racing Laboratory | Tang F.P.W.,Racing Laboratory | Wan T.S.M.,Racing Laboratory
Analytica Chimica Acta | Year: 2011

The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the original four LC/MS methods. The new method has better detection limits than the superseded methods. In addition, it covers new target analytes that could not be adequately detected by the four LC/MS methods. The new method involves solid-phase extraction (SPE) of two aliquots of equine urine using two Abs Elut Nexus cartridges. One aliquot of the urine sample is treated with β-glucuronidase before subjecting to SPE. A second aliquot of the same urine sample is processed directly using another SPE cartridge, so that drugs that are prone to decomposition during enzyme hydrolysis can be preserved. The combined eluate is analysed by UPLC/MS/MS using alternating positive and negative electrospray ionisation in the selected-reaction-monitoring mode. Exceptional chromatographic separation is achieved using an UPLC system equipped with a UPLC ® BEH C18 column (10cmL×2.1mm ID with 1.7μm particles). With this newly developed UPLC/MS/MS method, the simultaneous detection of 140 drugs at ppb to sub-ppb levels in equine urine can be achieved in less than 13min inclusive of post-run equilibration. Matrix interference for the selected transitions at the expected retention times is minimised by the excellent UPLC chromatographic separation. The method has been validated for recovery and precision, and is being used regularly in the authors' laboratory as an important component of the array of screening methods for doping control analyses of equine urine samples. © 2011 Elsevier B.V. Source

Wong J.K.Y.,Racing Laboratory | Tang F.P.W.,Racing Laboratory | Wan T.S.M.,Racing Laboratory
Drug Testing and Analysis | Year: 2011

The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of using homogenized horse liver instead of liver microsomes with the aim of saving preparation time and improving the correlation between in vitro and in vivo results. Indeed, the preparation of the homogenized horse liver was very simple, needing only to homogenize the required amount of liver. Even though no further purification steps were performed before the homogenized liver was used, the cleanliness of the extracts obtained, based on gas chromatography-mass spectrometry (GC-MS) analysis, was similar to that for liver microsomes. Herein, the results of the in vitro experiments carried out using homogenized horse liver for five anabolic steroids-turinabol, methenolone acetate, androst-4-ene-3,6,17-trione, testosterone, and epitestosterone-are discussed. In addition to the previously reported in vitro metabolites, some additional known in vivo metabolites in the equine could also be detected. As far as we know, this is the first report of the successful use of homogenized liver in the horse for carrying out in vitro metabolism experiments. © 2011 John Wiley & Sons, Ltd. Source

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