New anabolic steroid illegally used in cattle-structure elucidation of 19-norchlorotestosterone acetate metabolites in bovine urine.

4-Chloro-estr-4-en-17-ol-3-one, trivially named 19-norclostebol acetate or 4-chloro-19-nortestosterone acetate (NClTA), has been identified on the European black market in the late 1990s for possible use in breeding animals. After oral and subcutaneous administration of NClTA to bovine, urine samples were collected over a period of three weeks, and chemical structure of main excreted urinary metabolites was determined. After oral administration, the most abundant metabolites were mainly reduced as 4-chloro-19-norandrostan-3xi-ol-17-one and 4-chloro-19-norandrostan-3xi,17xi-diol. They were identified until 1 week after administration. Following subcutaneous injection, 4-chloro-19-norandrostan-3xi-ol-17-one was again of major abundance, but so were 4-chloro-19-norandrost-4-ene-3xi,17xi-diol and 4-chloro-19-norandrost-4-en-3xi-ol-17-one. They were detected at least 3 weeks after administration. Whatever the route of administration, metabolites were found mainly glucurono-conjugated; the only exception was metabolite 4-chloro-19-norandrostan-3xi-ol-17-one which was identified both in the sulpho- and glucurono-fractions.

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19-norandrosterone and 19-noretiocholanolone in human urine.

For the first time in the field of steroid residues in humans, demonstration of 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one) excretion in urine subsequent to boar consumption is reported. Three male volunteers agreed to consume 310 g of tissues from the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine samples before and during 24 h after meal intake. After deconjugation of phase II metabolites, purification and specific derivatisation of target metabolites, the urinary extracts were analysed by mass spectrometry. Identification was carried out using measurements obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-NE concentrations in urine reached 3.1 to 7.5 microg/L nearby 10 hours after boar tissue consumption. Levels returned to endogenous values 24 hours after. These two steroids are usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one), especially in the antidoping field. We have thus proved that eating tissues of non-castrated male pork (in which 17beta-nandrolone is present) might induce some false accusations of the abuse of nandrolone in antidoping.

Evidence for the presence of endogenous 19-norandrosterone in human urine.

In 1997, in the scope of antidoping control in sport, a not inconsiderable number of urine analysed by official laboratories revealed the presence of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one) metabolites: 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one). These repeated results on a short period of time generated some investigations and especially the verification of the possible production of these metabolites by an unknown endogenous route in adult entire male. Some experiences were led on different persons known to be non-treated with steroids and more precisely with nandrolone. Extractive methods were developed focusing on their selectivity, i.e. searching to eliminate at best matrix interferences from the target analytes. Gas chromatography coupled to mass spectrometry (quadrupole and magnetic instruments) was used to detect, identify and quantify the suspected signals. Two types of derivatization (TMS and TBDMS), a semi-preparative HPLC as well as co-chromatography proved unambiguously the presence, in more than 50% of the analysed urine (n = 40), of 19-NA at concentrations between 0.05 and 0.60 ng/ml. 19-NE was not detected with the developed methods (LOD<0.02 ng/ml). Experiments led on athletes showed that after a prolonged intense effort, the 19-NA concentration can be increased by a factor varying between 2 and 4. Even if some complementary researches have to be done in order to determine the maximal physiological level of 19-NA and 19-NE, these results should considerably change the strategy of antidoping laboratories.

4-Chlorotestosterone acetate metabolites in cattle after intramuscular and oral administrations.

The use of 4-chlorotestosterone acetate by farmers for cattle fattening was recently demonstrated although the use of this anabolic steroid is strictly forbidden in the European Union. We investigated the metabolism of 4-chlorotestosterone acetate in the bovine species after intramuscular and oral administration. Nineteen metabolites were detected in urine after intramuscular injection, and eight metabolites were identified. For this purpose, preparative HPLC, mass spectrometry with different ionization modes (electronic impact and chemical ionization), and different acquisition techniques were used (high resolution, selected ion monitoring, and scan measurement). Metabolite stereoisomerism was determined on the basis of retention time and organic synthesis. 4-Chloroepitestosterone (M2), 4-chloroandrost-4-en-3alpha-ol-17-one (M3), and 4-chloroandrost-4-ene-3,17-dione (M4) were identified as the main urinary markers of intramuscular administration. On the other hand, 4-chloroandrost-4-ene-3alpha,17beta-diol (M7), 4-chloroandrostan-3beta-ol-17-one (M5), and M2 were the primary indicators of an oral administration. In addition, we have shown that 95% of the metabolites were sulfo-conjugated, except for M3, which was partially conjugated to glucuronic acid. Finally, the main metabolites (M2, M3, and M4) were easily identified for 1.5 months after intramuscular administration.