Clive M. Pearce

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.

Analysis of methyloxime derivatives of intact esters of testosterone and boldenone in equine plasma using ultra high performance liquid chromatography tandem mass spectrometry

Analysis of equine plasma samples to detect the abuse of anabolic steroids can be complicated when the parent steroid is endogenous to the animal. Anabolic steroids are usually administered intramuscularly as synthetic esters and therefore detection of the exogenous esters provides unequivocal proof of illegal administration. An ultra high performance liquid chromatography tandem mass spectrometric (UPLC-MSMS) method for the analysis of esters of testosterone (propionate, phenylpropionate, isocaproate, and decanoate) and boldenone (undecylenate) in equine plasma has been developed. Esters were extracted from equine plasma using a mixture of hexane and ethyl acetate and treated with methoxyamine hydrochloride to form methyloxime derivatives. Metenolone enanthate was used as an internal standard. After chromatographic separation, the derivatized steroid esters were quantified using selected reaction monitoring (SRM). The limit of detection for all of the steroid esters, based on a signal to noise ratio (S/N) of 3:1, was 1-3 pg/mL. The lower limit of quantification (LLOQ) for the all of the steroid esters was 5 pg/mL when 2 mL of plasma was extracted. Recovery of the steroid esters was 85-97% for all esters except for testosterone decanoate which was recovered at 62%. The intra-day coefficient of variation (CV) for the analysis of plasma quality control (QC) samples was less than 9.2% at 40 pg/mL and less than 6.0% at 400 pg/mL. The developed assay was used to successfully confirm the presence of intact testosterone esters in equine plasma samples following intramuscular injection of Durateston® (mixed testosterone esters).

Detection of fluticasone propionate in horse plasma and urine following inhaled administration

Fluticasone propionate (FP) is an anti-inflammatory agent with topical and inhaled applications commonly used in the treatment of asthma in steroid-dependent individuals. The drug is used in racehorses to treat Inflammatory Airway Disease; this work was performed in order to advise on its use and detect potential misuse close to racing. Methods were developed for the extraction and analysis of FP from horse plasma and a carboxylic acid metabolite (FP-17βCOOH) from horse urine. The methods utilize ultra high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in order to detect the extremely low concentrations of analyte present in both matrices. The developed methods were used to analyse plasma and urine samples collected following inhaled administration of FP to six thoroughbred horses. FP was detected in plasma for a minimum of 72 h post-administration and FP-17βCOOH was detected in urine for approximately 18 h post-administration. The results show that it is possible to detect FP in the horse following inhaled administration.

Detection and pharmacokinetics of salbutamol in thoroughbred racehorses following inhaled administration

Salbutamol sulphate (Ventolin Evohaler) was administrated via the inhalation route to six horses at a dose of 0.5 mg every 4 h during the day for 2 days (total dose 4 mg). Urine and blood samples were taken up to 92 h postadministration. Hydrolyzed plasma and urine were extracted using solid phase extraction (SPE). A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification (LLOQ) for salbutamol of 10 pg/mL in plasma and urine. The parent drug was identified using UPLC-MS/MS. Most of the determined salbutamol plasma concentrations, post last administration, lie below the LLOQ of the method and so cannot be used for plasma PK analysis. Urine PK analysis suggests a half-life consistent with the pharmacological effect duration. An estimate of the urine average concentration at steady-state was collected by averaging the concentration measurements in the dosing period from -12 to 0 h relative to the last administered dose. The value was averaged across the six horses and used to estimate an effective urine concentration as a marker of effective lung concentration. The value estimated was 9.6 ng/mL and from this a number of detection times were calculated using a range of safety factors.