Metabolic Consequences of Chlorine Position in Testosterone Analogs: A GC-MS Study of C4- Versus C6-Chlorotestosterone Biotransformation.

The study investigates the metabolic profiles of 4-chlorotestosterone (clostebol, 4-CT) and 6α-chlorotestosterone (6-CT), highlighting how the chlorine atom's position may potentially influence the metabolism. In 4-CT, the chlorine atom is at C4, forming a stable vinyl halide; conversely, in 6-CT, the chlorine at C6 is an allylic halide, a much more reactive position. A single oral dose of 10 mg of 4-CT (n = 2) and 25 mg of 6-CT (n = 2) was administered, and urine samples were collected before and after the administration for 4 days. Samples were extracted with tert-butyl methyl ether at basic pH after enzymatic hydrolysis with β-glucuronidase. Trimethylsilyl derivatives were analyzed by mass spectrometry coupled to gas chromatography. Although apparently minor the difference between 4-CT and 6-CT was observed notable differences in the metabolic profiles due to the position of the chlorine atom that seems to define the metabolic route. Although the formation of the metabolite 4-chloro-androst-4-ene-3α-hydroxy-17-one (M1) was the main metabolic product for 4-CT, this is not the result for 6-CT. The Δ4-5 reduced metabolites for both 4-CT and 6-CT, identified as chlorinated androsterone and etiocholanolone, showed different excretion patterns, which may be linked to the inhibition degree of 5-reductases provoked by the chloride position. In the doping field, if the usual chromatographic conditions are used to detect steroids, special attention should be paid to the presence of a signal at the retention time of 4-CT M1 in a sample due to the similarities in transition relative ratio and elution time with the homologous 6-CT metabolite.