Veterinary Regulation and International Liaison

Changsha, China

Veterinary Regulation and International Liaison

Changsha, China
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Ho E.N.M.,Racing Laboratory | Kwok W.H.,Racing Laboratory | Lau M.Y.,Racing Laboratory | Wong A.S.Y.,Racing Laboratory | And 4 more authors.
Journal of Chromatography A | Year: 2012

A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment 17LKKTETQ23 is the active site within the protein thymosin β4 responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography-mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02ng/mL in equine plasma and 0.01ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses. © 2012 Elsevier B.V.


Wong A.S.Y.,Racing Laboratory | Ho E.N.M.,Racing Laboratory | Wan T.S.M.,Racing Laboratory | Lam K.K.H.,Veterinary Regulation and International Liaison | Stewart B.D.,Veterinary Regulation and International Liaison
Analytica Chimica Acta | Year: 2015

Oxyguno (4-chloro-17α-methyl-17β-hydroxy-androst-4-ene-3,11-dione) is a synthetic oral anabolic androgenic steroid commercially available without a prescription. Manufacturers of oxyguno claim that its anabolic effect in metabolic enhancement exceeds that of the classic anabolic steroid testosterone by seven times, but its androgenic side-effects are only twelve percent of testosterone. Like other anabolic androgenic steroids, oxyguno is prohibited in equine sports. The metabolism of oxyguno in either human or horse has not been reported and therefore little is known about its metabolic fate. This paper describes the in vitro and in vivo metabolic studies of oxyguno in racehorses with an objective to identify the most appropriate target metabolites for detecting oxyguno administration. In vitro studies of oxyguno were performed using horse liver microsomes. Metabolites in the incubation mixtures were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry in the EI mode after trimethylsilylation. In vitro metabolites identified include the stereoisomers of 4-chloro-17α-methyl-androst-4-ene-3-keto-11,17β-diol (M1a & M1b); 20-hydroxy-oxyguno (M2); and 4-chloro-17α-methyl-androst-4-ene-3-keto-11,17β,20-triol (M3). These novel metabolites were resulted from hydroxylation at C20, and/or reduction of the keto group at C11.For the in vivo studies, two geldings were each administered orally with a total dose of 210 mg oxyguno (52.5 mg twice daily for 2 days). Pre- and post-administration urine and blood samples were collected for analysis. The parent drug oxyguno was detected in both urine and blood, while numerous novel metabolites were detected in urine. The stereoisomers (M1a & M1b) observed in the in vitro studies were also detected in post-administration urine samples. Three other metabolites (M4 - M6) were detected. M4, 4-chloro-17α-methyl-androstane-11-keto-3,17β-diol, was resulted from reductions of the olefin group at C4 and the keto group at C3. M5 was resulted from hydroxylation at C20 and two reductions at either the olefin group at C4, the keto group at C3, or the keto group at C11. M6 was assigned as the 17-epimer of oxyguno. The major biotransformation pathways of oxyguno identified were reduction, hydroxylation and epimerisation. The structures of all metabolites were tentatively assigned by mass spectral interpretation. The longest detection time observed in urine was up to 10 h for the in vivo metabolite M4. Urinary and plasma oxyguno decreased rapidly and was no longer detectable at respectively 7 and 12 h post-administration. The above studies have provided useful information for the monitoring of oxyguno administration in racehorses. © 2015 Elsevier B.V.


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.


Leung G.N.W.,Racing Laboratory | Kwok W.H.,Racing Laboratory | Wan T.S.M.,Racing Laboratory | Lam K.K.H.,Veterinary Regulation and International Liaison | Schiff P.J.,Veterinary Regulation and International Liaison
Drug Testing and Analysis | Year: 2013

Formestane (4-hydroxyandrost-4-ene-3,17-dione) is an irreversible steroidal aromatase inhibitor with reported abuse in human sports. In 2011, our laboratory identified the presence of formestane in a horse urine sample from an overseas jurisdiction. This was the first reported case of formestane in a racehorse. The metabolism of formestane in humans has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the in vitro and in vivo metabolic studies of formestane in horses, with the objective of identifying the target metabolite with the longest detection time for controlling formestane abuse. In vitro metabolic studies of formestane were performed using homogenized horse liver. Seven in vitro metabolites, namely 4-hydroxytestosterone (M1), 3β,4α-dihydroxy-5β-androstan-17-one (M2a), 3β,4β-dihydroxy-5β-androstan-17-one (M2b), 3β,4α-dihydroxy-5α-androstan-17-one (M2c), androst-4-ene-3α,4,17β-triol (M3a), androst-4-ene-3β,4,17β-triol (M3b), and 5β-androstane-3β,4β,17β-triol (M4) were identified. For the in vivo studies, two thoroughbred geldings were each administered with 800 mg of formestane (32 capsules of Formadex) by stomach tubing. The results revealed that the parent drug and seven metabolites were detected in post-administration urine. The six in vitro metabolites (M1, M2a, M2b, M2c, M3a, and M3b) identified earlier were all detected in post-administration urine samples. In addition, 3α,4α-dihydroxy-5α-androstan-17-one (M2d), a stereoisomer of M2a/M2b/M2c, was also identified. This study has shown that the detection of formestane administration would be best achieved by monitoring 4-hydroxytestosterone (M1) in the glucuronide-conjugated fraction. M1 could be detected for up to 34 h post-administration. In blood samples, the parent drug could be detected for up to 34 h post administration. © 2013 John Wiley & Sons, Ltd.

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