UPLC–ESI-Q-TOF-MSE identification of urinary metabolites of the emerging sport nutrition supplement methoxyisoflavone in human subjects

• Methoxyisoflavone is an isoflavone used by bodybuilders for its ergogenic properties.
• Five metabolites, resulting from O-demethylation and hydroxylation, are identified.
• 5-Methyl-7-hydroxyisoflavone is the major metabolite.
• Mass spectra reveal specific double CO loss and 1,3 retro Diels-Alder fragmentation.

Methoxyisoflavone (5-methyl-7-methoxyisoflavone) is a synthetic isoflavone used by bodybuilders for its ergogenic properties. A recent study demonstrated that methoxyisoflavone metabolites can induce false-positive results in urinary immunoassay screening tests for cannabinoids, and only one metabolite has been identified. To improve the knowledge on the metabolic pathways of methoxyisoflavone, ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF) was applied. Urine samples were obtained from methoxyisoflavone regular users. After enzymatic hydrolysis and liquid–liquid extraction, the samples were analyzed by UPLC–Q-TOF fitted with an electrospray ionization source (ESI) operating under positive ion mode. Mass data were acquired with the MSE method. Five metabolites were identified. Those were divided into two metabolic pathways, depending on whether the B ring hydroxylation was preceded or not by the O-demethylation of the methoxy group. The MSE mass spectra of methoxyisoflavone and its metabolites are specific of isoflavones structures and revealed 1,3 retro Diels-Alder fragmentation and double CO loss. Losses of small neutral molecules CO and H2O, and radical CH3, typical of flavonoids, were also observed. This study illustrates the capacity of the sensitive UPLC–Q-TOF analytical system, combined with the MSE method of collection of fragmentation data, to rapidly elucidate the unknown xenobiotics metabolism.

Phase I metabolic pathways of methoxyisoflavone (5-methyl-7-methoxyisoflavone).Figure optionsDownload as PowerPoint slide