Anabolic steroids
In human sport, anabolic steroids are used as body-building drugs, sometimes in very large quantities, in events such as weightlifting and shot-putting. In horseracing, their use seems to be more general. They are said to improve a horse’s appetite and to produce behavioural changes of a masculinising type in geldings and mares, though they may also be used in stallions. There are broadly two chemical types based upon the androstane and estrange ring systems: those with a 17a-alkyl group, which are active orally but possess hepatotoxicity, and 19-nor derivatives administered by injection. Although steroidal oestrogens and some stilbenes reputedly possess anabolic activity and are employed in beef production, they do not appear to be used as doping agents. Studies (Houghton 1977; Houghton and Dumasia 1979, 1980; Dumasia and Houghton 1981, 1984; Dumasia et al. 1983) have shown the metabolism of the veterinary anabolic agents nandrolone, testosterone, boldenone and trenbolone in the horse to be complex, with both phase I and phase II processes playing import ant roles. Nandrolone has been shown to be endogenous to the male horse (Houghton et al. 1984) and this, and testosterone, are controlled by threshold values (see Table 9.2). Metabolism of 17a-alkyl anabolic agents has also been studied in the horse (Schoene et al. 1994; McKinney et al. 2001). These studies have thus been essential to identify key analytes for the development of screening and confirmatory analysis methods.
Radioimmunoassay and subsequently ELISA procedures have been developed for these veterinary anabolic agents (Jondorf 1977; Jondorf and Moss 1977) and commercial assays are currently available. Also, the identification of the major metabolites for these steroids has enabled the development of multi-residue GC MS screening procedures (Dumasia et al. 1986; Houghton et al. 1986). The metabolism differs markedly from that in humans, with the reduction of the 4-ene-3-one group and epimerization at C17 being major phase I pathways. Phase II processes also differ markedly, with sulfate conjugation playing an important role in horses, but a minor role in humans. Thus, the developed methods for screening and confirmatory analysis by mass spectrometric methods require steps for cleavage of both glucuronic acid and sulfate conjugates.
The metabolism of anabolic steroids in humans has been documented, especially by Schanzer and co-workers (e.g. Schanzer and Donike 1993; Schanzer 1996). Synthetic anabolic steroids used in humans may be detected readily by the use of GC-MS analysis. Detection of the administration of pseudo-endogenous compounds (i.e. compounds that are virtually identical to those of endogenous origin, such as testosterone), present a more complex problem although the use of CIRMS (see above) has greatly assisted obtaining evidence to prove administration. There are several methods to indicate that testosterone has been administered to a male human. Natural testosterone production is controlled by a feedback mechanism that involves the pituitary gland, and administration of testosterone suppresses the natural production of pituitary hormones, such as luteinising hormone (LH) and follicle stimulating hormone (FSH). GC-MS analysis may be used to measure the testosterone concentration in urine, and immunoprocedures to determine LH and hence the urinary ratio of total (free plus conjugated) testosterone to LH. Testosterone is measured in nmol/L, and LH in International Units of Human Menopausal Gonadotropin 2nd International Reference Preparation per litre (HMG-IR2/L). A ratio in excess of 200 is abnormal (Brooks et al. 1979).
In female humans, whose pituitary hormones may be suppressed by the use of oral contraceptives, the testosterone to LH ratio is of little evidential value. Instead, a method suitable for both males and females is to measure the ratio of total testosterone to epitestosterone using GC-MS. In this method, the bis(trimethylsilyl) derivative is formed and the intensity of the molecular ions at m/z 432, under electron impact conditions for both of the steroid derivatives, is used to determine the ratio (Donike and Zimmermann 1980). The internationally accepted limit for the ratio is 6, although WADA currently requires reports to be issued whenever a ratio of 4 is reached. A method to detect the administration of dihydrotestosterone, the more active metabolite of testosterone, has also been proposed (Kicman et al. 1995) based on the perturbation of the endogenous hormone profile following administration. Isotope ratio MS is also now used to assist detection of the administration of testosterone and related compounds. Sampling carried out only at the time of competition has enabled the misuser to switch from using a preferred synthetic anabolic steroid to testosterone or to a synthetic anabolic steroid that is eliminated rapidly in sufficient time to escape detection. Thus sampling out-of competition and with minimum notice as well as at competitions is now accepted as the optimal approach to detecting, and thereby limiting, the use of anabolic steroids in humans. Mass spectral and chromatographic data for many anabolic steroids and their metabolites have been published by Ayotte et al. (1996). Kicman and Gower (2003) have reviewed the use of anabolic steroids in sports, including analytical perspectives.
