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

Abstract

ABSTRACT 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.