Identification and synthesis of (Z)‐3′‐hydroxy clomiphene as a new potential doping‐relevant metabolite of clomiphene

Abstract

A recent study addressed the possibility of unintentional ingestion of clomiphene through residues in chicken eggs. The method developed here helped distinguish between microdose intake of (E/Z)‐clomiphene citrate and consumption of clomiphene‐containing eggs by the urinary pattern of four mono‐hydroxylated clomiphene metabolites. However, reanalyses of doping‐control samples, which showed an adverse analytical finding for clomiphene, revealed a hydroxy clomiphene (HC) isomer that was not found after microdose intake or after consumption of clomiphene‐containing eggs and could not be assigned to any of the available reference compounds. The aim of the present follow‐up study was to identify this HC isomer and to characterize this metabolite with respect to its potential properties as long‐term metabolite in doping controls.Methods(E/Z)‐3′‐HC and (E/Z)‐4′‐HC were synthesized involving the McMurry reaction. An ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and optimized after a derivatization step with dansyl chloride to separate eight HC isomers. Using this method, urine samples from a controlled clomiphene administration study were analyzed, in which male study participants received therapeutic doses of clomiphene for 30 days and collected urine samples for up to 8 months. Thus, isomer‐specific HC elimination profiles could be monitored.ResultsThe metabolite previously found in doping‐control samples was identified as (Z)‐3′‐HC. The elimination profiles of the different HCs confirmed previous results, with (Z)‐3‐HC being the most abundant urinary hydroxy metabolite shortly after administration. A new finding was that the data suggest that (Z)‐3′‐HC is excreted at higher relative concentrations only several weeks after drug intake.ConclusionThese findings might be of particular importance in sport drug testing as they can assist in the decision‐making process to distinguish between intentional doping and inadvertent exposure to clomiphene via food contamination.