Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the hair strand or segment with methanol/water (1:1), milling, extraction of the hair material in an ultrasonic bath using methanol, purification by liquid-liquid extraction (n-pentane/methanol, 25:1) and HPLC cleanup, derivatisation of the relevant LC fractions with MSTFA, and measurement using GC-MS/MS technique. The first objective of our study was the detection of exogenous nandrolone (nortestosterone, NT) in the horse hair; therefore nandrolone-associated compounds [nandrolone dodecanoate administered intramuscularly (i.m.) and a mixture of 4-estrenediol and 4-estrenedione, transdermal] were administered to four geldings. The highest concentrations of NT following i.m. treatment were measured after 10 days in a 2-cm hair segment (up to 18 pg/mg); NT was detectable for up to 120 days and in some cases up to 330 days in tail hair (limit of detection 0.3 pg/mg). Following transdermal application, nandrolone as well as the administered prohormones were identified in tail and mane until the latest sampling at 3 months. Furthermore, untreated stallions (128) were investigated to estimate the range of endogenous levels of NT and testosterone (T) in hair. Maximum values of 3 pg/mg (NT) and 1 pg/mg (T) were quantified originating from endogenous formation in the male horse. Additionally, a possible relationship between steroid concentrations in hair specimens and the age of stallions was appraised. NT and T were not detected in hair samples of control geldings. Following nandrolone treatment of geldings, highest values in hair exceeded the endogenous amount detected in untreated stallions. Therefore comparison of concentrations measured in control samples with the estimated endogenous levels could give a clue to exogenous application in cases of abnormally high amounts of NT or T. The possibility of the evaluation of threshold values is discussed as a means to verify an exogenous administration of NT and T in hair samples. Furthermore, the detection of a synthetic substance in hair, e. g. the parent steroid ester by itself, would be unequivocal proof of an exogenous origin of NT or T and the previous medication of the stallion.

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Doping control of anabolic substances is normally carried out with urine samples taken from athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair material is preferred in cases of limited availability or complicated collection of urine samples, e.g. from horses.In this work, possible ways of interpretation of analytical results in hair samples are discussed and illustrated by practical experiences. The results demonstrate the applicability of hair analysis to detect anabolic steroids and also to obtain further information about previous abuse. Moreover, the process of incorporation of steroids into hairs is described and the consequences on interpretation are discussed, e.g. on the retrospective estimation of the application date.The chosen examples deal with the detection of the anabolic agent testosterone propionate. Hair samples of an application study, as well as a control sample taken from a racing horse, were referred to. Hair material was investigated by a screening procedure including testosterone, nandrolone and several esters (testosterone propionate, phenylpropionate, decanoate, undecanoate, cypionate; nandrolone decanoate, dodecanoate and phenylpropionate; limits of detection (LODs) between 0.1 and 5.0 pg/mg). Confirmation of testosterone propionate (LOD 0.1 pg/mg) was carried out by an optimised sample preparation. Trimethylsilyl (TMS) and tert-butyl dimethylsilyl derivatives were detected by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).

The potential influence of alcohol consumption on endogenous steroids has already been described in the literature. In those studies the ethanol level after ingestion was monitored using its concentration in blood, urine or saliva. Corresponding methods are not commonly used in anti-doping laboratories. Ethylglucuronide (EtG), which can be easily detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), appears to be a more suitable parameter for this purpose. It is slowly excreted into the urine and indicates alcohol intake for a much longer period than blood or urinary alcohol and it is therefore routinely used for legal purposes as an alcohol consumption marker. In pharmacokinetic studies that aimed to establish calculation models after ethanol intake, the formation of EtG was observed to coincide with elevated urinary testosterone/epitestosterone (T/E) ratios. Similarly, large amounts of EtG were correlated with abnormal steroid profiles found in routine doping samples. In this pilot study, several cases with significantly elevated T/E ratios were associated with urinary EtG concentrations higher than 50 microg/mL. These findings confirmed recent intake of ethanol in considerable amounts and suggest a connection to changes in specific steroid profile parameters. Owing to the ease with which procedures to determine EtG can be carried out, and the potential for such procedures to be introduced into screening schemes, the inclusion of this marker in the final evaluation of suspicious outliers in T/E ratio longitudinal studies would seem to be very useful.

Summary Reasons for performing study: The β2‐agonist clenbuterol is commonly administered for therapeutic purposes in the horse, but its use an an anabolic agent is illegal. Clenbuterol can be detected in blood and urine for a relatively short period after administration and detection in hair could enhance the analytical range and be used to determine the history of clenbuterol application. Hypothesis: That detection in mane or tail hair is possible over an extended period. Methods: Four horses received 0.8 μg elenbuterol hydrochloride/kg bwt b.i.d. for 10 days. Four other horses were used as untreated controls. Blood, urine, mane and tail hair samples were taken on Day 0 (before) and 5, 10, 30, 35, 40, 60, 90, 120, 150 and 360 days after start of treatment. Gas chromotography/high resolution mass spectrometry (GC/HRMS) was developed for clenbuterol analysis: limit of detection was 0.2 pg/mg; intra‐assay repeatability limit r = 0.06 (confidence level 95%); interassay repeatability limit r = 0.03 (confidence level 95%). Prior to treatment, clenbuterol was absent from all samples analysed. Results: Clenbuterol was detectable as early as Day 5 in tail and mane hair of Segment 1 (0–20 mm from the roots) and was maximal on Day 90. However, as time progressed, shift into lower 20 mm segments was observed. On Day 360, the maximum concentration (up to 21 pg/mg) was located in Segment 13, i.e. 26–28 cm from roots of hair. Clenbuterol was not detectable in blood or urine after Day 30. Mane and tail hair results were very similar. Conclusions: The study showed that the β‐agonist clenbuterol can be found in mane and tail hair of horses after extended periods. Potential relevance: It will be possible to detect clenbuterol in breeding and show horses where anabolic drugs have been used illegally to improve conformation. This method may also be helpful to monitor therapeutic clenbuterol treatment.;