Metabolism of formestane in humans: Identification of urinary biomarkers for antidoping analysis.

Formestane (4-hydroxyandrost-4-ene-3,17-dione, 4OH-AED) is an aromatase inhibitor prohibited in sports. In recent years, it has been demonstrated that it can also originate endogenously by the hydroxylation in C4 position of androstenedione. Thus, the use of isotope ratio mass spectrometry (IRMS) is mandatory according to the World Antidoping Agency (WADA) to discriminate endogenous from synthetic origin. In a previous work and after oral administrations of formestane (4OH-AED), the ratio between the main formestane metabolite (4α-hydroxyepiandrosterone; 4OH-EA) and formestane parent compound could help to identify the endogenous origin, avoiding unnecessary and costly IRMS confirmations. In the present work, we investigated whether the same criteria could also be applied after transdermal applications. Six volunteers were transdermally treated once with formestane. Urine samples were collected for 120 h postadministration and analyzed by gas chromatography coupled to mass spectrometry (GC-MS and GC-MS/MS). Formestane and its major metabolites were monitored. The kinetic profile of formestane and its main metabolites was found different between oral and transdermal application. A shift on the excretion of the metabolites compared to formestane itself that can be observed after the oral administration, is absent after the transdermal one. This makes that a simple criteria cannot be applied to differentiate the endogenous from the synthetic origin based on metabolic ratios. The ratio between 4-hydroxyepiandrosterone and 4-hydroxyandrosterone (4OH-A) can be used to differentiate the route of administration. Ratios higher than one (4OH-EA/4OH-A > 1) are diagnostic of an oral administration. This allows to correctly interpret the 4OH-EA/4OH-AED ratio as proposed in our previous investigation. The results of this work demonstrate that the use of appropriate biomarkers (metabolic ratios) helps to reach correct conclusions without using complex and costly instrumentation approaches.

Reference ranges for the urinary steroid profile in a Latin-American population.

The urinary steroid profile has been used in clinical endocrinology for the early detection of enzyme deficiencies. In the field of doping, its evaluation in urine samples is used to diagnose the abuse of substances prohibited in sport. This profile is influenced by sex, age, exercise, diet, and ethnicity, among others; laboratories own reference ranges might compensate for ethnic differences among population and inter-laboratory biases. This paper shows the reference ranges obtained in the Antidoping Laboratory of Havana for the following steroid profile parameters: ten androgens (testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstan-3α,17ß-diol, 5ß-androstan-3α,17ß-diol, dehydroepiandrosterone, epiandrosterone, 11ß-hydroxyandrosterone and 11ß-hydroxyetiocholanolone), three estrogens (estradiol, estriol and estrone), two pregnanes (pregnanediol and pregnanetriol) and two corticosteroids (cortisol and tetrahydrocortisol). The urine samples (male: n = 2454 and female: n = 1181) and data obtained are representative of population from Latin-American countries like Cuba, Venezuela, Mexico, Dominican Republic, Guatemala and Chile. Urine samples were prepared by solid-phase extraction followed by enzymatic hydrolysis and liquid-liquid extraction with an organic solvent in basic conditions. Trimethylsilyl derivatives were analyzed by gas chromatography coupled to mass spectrometry. Reference ranges were established for each sex, allowing the determination of abnormal profiles as a first diagnostic tool for the detection of the abuse of androgenic anabolic steroids. The comparison with the Caucasian population confirms that the urinary steroid profile is influenced by ethnicity.