Detection of urinary arimistane metabolites in humans using liquid chromatography–mass spectrometry: Complementary results to gas chromatography mass spectrometric data and its application to antidoping analyses

Abstract

Rationale The metabolism of arimistane (Arim) was first described in 2015, and androst‐3,5‐diene‐7β‐ol‐17‐one was proposed as the main metabolite excreted in urine. Recently, a more detailed study describing the findings in urine after the administration of Arim has been published. This study corroborated the previously described metabolite but also described several phase I and II metabolites, analyzing trimethylsilylated urinary extracts using accurate mass spectrometry coupled to gas chromatography (GC/qTOF). The present communication is an extension of this late investigation aiming to implement the results of Arim metabolism using either accurate mass spectrometry and/or triple quadrupole tandem mass spectrometry, both coupled to liquid chromatography (LC/qTOF and LC/QqQ). Methods The samples used in this study were the same as previously studied using GC/qTOF. One single oral dose of Arim was administered to three volunteers, and samples collected before and up to 10 h after the Arim administration were analyzed. The unconjugated fraction of urine was removed, and the hydrolysis was performed with β‐glucuronidase from Escherichia coli. The extracts were reconstituted in water:acetonitrile before the LC/qTOF and LC/QqQ analysis. Results The presence of the proposed metabolites studied using GC was verified by accurate mass measurements. Twelve metabolites not found in the blank urine samples were identified by the accurate mass spectra with acceptable errors between −7.5 and 8.1 ppm: 4 reduced metabolites, 4 monohydroxylated metabolites, and 4 with an additional hydroxylation (bis‐hydroxylated metabolites). Unlike in the study carried out using GC/qTOF, Arim itself was found in the samples of the three volunteers. Conclusions Twelve metabolites were identified, and specific transitions were proposed. Despite the good results, some limitations remain. As for GC/qTOF, the α‐ or β configuration of hydroxy groups, as well as the exact position for some unsaturation, cannot be assigned with certainty. Because certified reference materials of these metabolites are not yet available, the molecular structures were hypothesized considering the previous study using GC.

Annual banned‐substance review: Analytical approaches in human sports drug testing 2020/2021

“…In that context, Kollmeier et al 92 In contrast to formestane, arimistane is not known to be of natural/endogenous origin. Hence, the identification of arimistane in urine could be considered as proof of drug use, and analyzing arimistane and its main metabolite androst-3,5-dien-7β-ol-17-one by LC-MS/ MS has been shown to provide superior sensitivity compared to GC-MS(/MS) as reported by Martinez-Brito et al 93 Using postadministration samples collected from three study participants after the oral administration of 25 mg of arimistane, the intact drug was detected up to 10 h (later samples were not analyzed). Of note, the sample preparation included an initial LLE to remove unconjugated analytes followed by an enzymatic hydrolysis and another LLE, indicating that the observed arimistane was glucurono-conjugated despite the lack of a hydroxyl function.…”

Variation of Sequential Ligandrol (LGD-4033) Metabolite Levels in Routine Anti-Doping Urine Samples Detected with or without Other Xenobiotics

“…Estimated Concentration (ng/mL) Sample 1 Arimistane (Androst-3,5-diene-7,17-dione) [15] 110.65 Arimistane M (Androst-3,5-diene-7β-ol-17-one) [16] 454.38…”