Development and validation of an LC-MS/MS method for testosterone, 4-androstene-3,17-dione and pregnenolone in human serum

CBF 2.0 2025 -- Biomarkers are measurable indicators of biological processes, disease states or responses to stimuli. Steroid hormones, a crucial class of biomarkers, play vital roles in reproductive function, metabolic homeostasis and adrenal activity. Accurate quantification of testosterone, 4-androstene-3,17-dione and pregnenolone in biological matrices is essential for endocrine disorder investigations, disease monitoring and pharmacodynamic studies. However, quantitative bioanalysis of these endogenous steroid hormones presents significant challenges: low physiological concentrations (pg/mL levels), high structural similarity among steroids causing interferences, complex biological matrices and high baseline noise. To address these challenges, we developed and validated a robust method combining supported liquid extraction with LC-MS/MS for simultaneous quantification of these three steroid hormones, providing a reliable tool for clinical and research applications.