New Potential Biomarker for Methasterone Misuse in Human Urine by Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry.

Jianli Zhang,Xiaobing Wang,Yan Wang,Yun Wu,Jianghai Lu,Youxuan Xu,Yinong Zhang

doi:10.3390/ijms17101628

Abstract

In this study, methasterone urinary metabolic profiles were investigated by liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS) in full scan and targeted MS/MS modes with accurate mass measurement. A healthy male volunteer was asked to take the drug and liquid–liquid extraction was employed to process urine samples. Chromatographic peaks for potential metabolites were hunted out with the theoretical [M − H]− as a target ion in a full scan experiment and actual deprotonated ions were studied in targeted MS/MS experiment. Fifteen metabolites including two new sulfates (S1 and S2), three glucuronide conjugates (G2, G6 and G7), and three free metabolites (M2, M4 and M6) were detected for methasterone. Three metabolites involving G4, G5 and M5 were obtained for the first time in human urine samples. Owing to the absence of helpful fragments to elucidate the steroid ring structure of methasterone phase II metabolites, gas chromatography mass spectrometry (GC-MS) was employed to obtain structural information of the trimethylsilylated phase I metabolite released after enzymatic hydrolysis and the potential structure was inferred using a combined MS method. Metabolite detection times were also analyzed and G2 (18-nor-17β-hydroxymethyl-2α, 17α-dimethyl-androst-13-en-3α-ol-ξ-O-glucuronide) was thought to be new potential biomarker for methasterone misuse which can be detected up to 10 days.

Highlights

Methasterone is an orally active anabolic agent exhibiting androgenic activity [1,2]
The objective of this paper is to identify and characterize intact methasterone metabolites by liquid chromatography coupled with quadrupole time-of-flight mass spectrometric technique (LC-QTOF-MS) and gas chromatography mass spectrometry and find biomarkers to improve our routine analysis in doping control
The nine phase II metabolites were detected with accurate mass measurement by LC-QTOF-MS instrumentation, and a LC cleanup procedure was employed to isolate these metabolites and the LC-fraction containing relative conjugated metabolites was subjected to enzymatic hydrolysis and TMS derivatization

Summary

Introduction

Methasterone (superdrol, and methyldrostanolone) is an orally active anabolic agent exhibiting androgenic activity [1,2]. Methasterone and its main metabolite (dihydromethasterone) in free and glucuronide fractions were detected in human urine by GC-MS after taking the nutritional supplement containing 10 mg methasterone and the amount of dihydromethasterone was five times greater than that of parent drug [14]. Two biomarkers, parent drug and its C3-reduced metabolite, were employed for monitoring methasterone misuse and an enzymatic hydrolysis step was involved prior to GC-MS analysis in doping control field, which was time-consuming and indirect [15]. The objective of this paper is to identify and characterize intact methasterone metabolites (mainly sulfate and glucuronide conjugates) by liquid chromatography coupled with quadrupole time-of-flight mass spectrometric technique (LC-QTOF-MS) and gas chromatography mass spectrometry and find biomarkers to improve our routine analysis in doping control

Objectives

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Discussion

Conclusion