Comprehensive steroid profiling by liquid chromatography coupled to high resolution mass spectrometry

Area of Science:

• Analytical Chemistry
• Biochemistry
• Metabolomics

Background:

• Steroid hormone analysis is crucial for understanding physiological processes and detecting exogenous substances.
• Comprehensive profiling of urinary steroids, including both free and conjugated forms, presents analytical challenges.

Purpose of the Study:

• To develop and validate a steroidomics workflow for the simultaneous analysis of a wide range of free and conjugated steroids in urine.
• To create a comprehensive database for steroid identification and characterization.
• To assess the workflow's utility in detecting metabolic changes induced by anabolic treatments.

Main Methods:

• Solid-phase extraction (SPE) optimization for sample preparation.
• Ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) for simultaneous analysis.
• Development of a homemade database (>150 steroids) for metabolite identification.
• In vivo study involving anabolic treatment (boldenone undecylenate) in veal calves.

Main Results:

• The workflow demonstrated high selectivity, repeatability (<13%), and linearity (R2>0.985) for 44 representative steroids.
• Successfully identified known phase II metabolites of boldenone (glucuronide and sulfate) and observed changes in epiboldenone glucuronide.
• Detected 31 unidentified steroid signals impacted by the exogenous treatment.
• First study to simultaneously profile free and conjugated C18, C19, and C21 steroid hormones using UHPLC-HRMS/MS.

Conclusions:

• The developed steroidomics workflow provides a comprehensive approach for analyzing urinary steroid profiles.
• The method is effective in identifying known and potential novel steroid biomarkers, even after exogenous administration.
• This workflow offers a powerful tool for doping control, metabolic research, and understanding steroid homeostasis.