Investigations into the feasibility of routine ultra high performance liquid chromatography-tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids, anabolic steroid esters and related compounds.

The detection of the abuse of anabolic steroids in equine sport is complicated by the endogenous nature of some of the abused steroids, such as testosterone and nandrolone. These steroids are commonly administered as intramuscular injections of esterified forms of the steroid, which prolongs their effects and improves bioavailability over oral dosing. The successful detection of an intact anabolic steroid ester therefore provides unequivocal proof of an illegal administration, as esterified forms are not found endogenously. Detection of intact anabolic steroid esters is possible in plasma samples but not, to date, in the traditional doping control matrix of urine. The analysis of equine mane hair for the detection of anabolic steroid esters has the potential to greatly extend the time period over which detection of abuse can be monitored. Equine mane hair samples were incubated in 0.1M phosphate buffer (pH 9.5) before anabolic steroids (testosterone, nandrolone, boldenone, trenbolone and stanozolol), anabolic steroid esters (esters of testosterone, nandrolone, boldenone and trenbolone) and associated compounds (fluticasone propionate and esters of hydroxyprogesterone) were extracted by liquid-liquid extraction with a mix of hexane and ethyl acetate (7:3, v:v). Further sample clean up by solid phase extraction was followed by derivatisation with methoxylamine HCL and analysis by UHPLC-MS/MS. Initial method development was performed on a representative suite of four testosterone esters (propionate, phenylpropionate, isocaproate and decanoate) and the method was later extended to include a further 18 compounds. The applicability of the method was demonstrated by the analysis of mane hair samples collected following the intramuscular administration of 500 mg of Durateston(®) (mixed testosterone esters) to a Thoroughbred mare (560 kg). The method was subsequently used to successfully detect boldenone undecylenate and stanozolol in hair samples collected following suspicious screening findings from post-race urine samples. The use of segmental analysis to potentially provide additional information on the timing of administration was also investigated.