Highly specific ID-UHPLC-MS/MS method for analyzing polar and non-polar steroid hormones

The accurate and precise measurement of endogenous steroid hormone levels in serum is essential for their use as biomarkers of endocrine and metabolic diseases in patient care and translational science. Herein, we describe a newly developed, highly accurate, and precise isotope-dilution liquid chromatography-mass spectrometry (LC-MS/MS) method capable of simultaneously measuring eight clinically relevant polar and non-polar steroid hormones in 200 µL of serum. This steroid hormone panel method uses sequential liquid-liquid extractions for the isolation of total testosterone (TT), estradiol (E2), progesterone (P4), 17-hydroxyprogesterone (17-OHP), androstenedione (AD), estrone (E1), estrone sulfate (E1S), and dehydroepiandrosterone sulfate (DHEAS) without derivatization or hydrolysis. The method demonstrated a broad analytical measurement range for all eight hormones as a result of improved selectivity and sensitivity, making it suitable for the analysis of general population serum samples, including postmenopausal women and children. The total imprecision, expressed as coefficients of variation, was evaluated at three levels for each analyte and ranged from 3.5 to 10.7%. The mean bias to well-established secondary reference materials was -1.00% for TT and 1.91% for E2. When applied to 268 commercially sourced serum samples from men, women, and children, only 2% of measurement results were below the limits of detection; therefore, this method was deemed suitable for measurement of eight steroid hormones across the general population. The establishment of this new steroid hormone panel LC-MS/MS method will facilitate investigations of associations between health disorders and individualized steroid profiles, which can be utilized for evaluations at the individual or population levels.