Presence of ß2 -agonist growth promoters in human urine samples: GC-MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses.

ß2 -adrenergic agonists having the potential to be misused to enhance performance for their thermogenic and anabolic properties are prohibited in sports. Clenbuterol, ractopamine and zilpaterol are utilised legally or illegally as growth promoters of animals raised for their meat. No withdrawal times are imposed for ractopamine prior to slaughter; residues are detected in meat of treated animals, which constitutes a risk of inadvertent consumption. Insufficient information is available on the fate of ractopamine in humans to implement efficient detection in athletes' urine samples. We have developed a confirmation procedure for total ractopamine in urine following the enzymatic hydrolysis of glucuronides and sulphates and the conversion to tri-TMS derivative (limit of identification at 0.15 ng/ml). The sulphates were found to form between 85% to 97% of ractopamine excreted in athletes' urine samples analysed routinely or in volunteers following the administration of a micro-dose of 2.5 µg. Peak levels were reached at 2 to 6 h and decreased rapidly below 1 ng/ml 10 h after dosing. With one exception, the highest level estimated in athletes' samples was 1.2 ng/ml. Zilpaterol was confirmed in a few urine samples collected in the USA and Mexico (highest level 2 ng/ml), while hundreds of athletes' samples were reported to contain clenbuterol by our laboratory over the past 7 years. Most of these cases originated from Mexico (n = 102) and Guatemala (n = 119), often clustered in events during which multiple samples were collected, and for the vast majority, in levels lower than 0.2 ng/ml.

Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR.

Two steroids were identified in a supplement named D-2 following the detection of unknown compounds during the routine testing of an athlete's sample. The main glucuroconjugated metabolites were isolated from this urine by high performance liquid chromatography (HPLC) following enzymatic hydrolysis and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses as being 2α-hydroxy-5α-androst-3-en-17-one (M1) and 2ß,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4ß-dihydroxy-5α-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5α-androst-2-en-17-one (1) and 5α-androst-3-en-17-one (2) were present in the first D-2 products offered on the Internet. Later, the corresponding 17-hydroxyl compounds were offered as such or as esters (acetate, cypionate) in different relative ratios. Both M2 and M3 were synthesized from the trans-diaxial hydrolysis of the corresponding 2α,3α- and 3α,4α-epoxides (3). These were excreted in the hours following the controlled administration of the commercial product called D-2 R to a male volunteer and were also produced from the incubation of 1 and 2 with S9 liver fractions. Some preparations contain predominantly the alkene in C-2 and, therefore, an efficient detection method must include both primary metabolites M1 and M2. The latter was found equally in the fractions extracted following the enzymatic hydrolysis with ß-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd.