An investigation on the metabolic pathways of synthetic isoflavones by gas chromatography coupled to high accuracy mass spectrometry

Abstract

Rationale:Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens. Methods:The in vivo metabolism of methoxyisoflavone (5-methyl-7methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS).Results: Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation. Conclusions:When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis.

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…Among those, established target metabolites such as androst‐3,5‐diene‐7β‐ol‐17‐one were found but also newly identified features attributed to C‐2‐hydroxylated species such as androst‐5‐ene‐2α/β‐ol‐7,17‐dione, based on mass spectrometric information. With a single oral dose of 25 mg of arimistane, no significant effect on the tested persons' steroid profiles was observed; whether or not the continued use of synthetic isoflavones as “natural” aromatase inhibitors might necessitate consideration as confounding factors of the steroidal module of the ABP was discussed by Iannone et al 143 In order to enable anti‐doping laboratories to identify the administration of isoflavones and, if required, include the presence of these into ABP interpretations, elimination studies with methoxyisoflavone (450 mg per day, 3 days) and ipriflavone (200 mg per day, 3 days) were conducted, yielding eight and six tentatively identified metabolites, respectively. The most common metabolic reactions were dealkylation and hydroxylation, allowing for an unequivocal detection of isoflavone administration.…”