LC-MS/MS-based simultaneous high-sensitivity quantification of clinically relevant high-potency androgens and estrogens in plasma and serum.

We present a robust assay coupling liquid chromatography with tandem mass spectrometry (LC-MS/MS) that enables the quantification of 19 steroids with high sensitivity in 300 μL of human plasma or serum. It includes a broad array of classical adrenal precursors, progesterone, estrogens, androgens, and their metabolites - dehydroepiandrosterone (DHEA), androstenediol (5-diol), androstenedione (A4), dihydrotestosterone (DHT), testosterone (T), androsterone (AST), 5α-androstan-3α-17β-diol (3α-diol), 5α-androstan-3β-17β-diol (3β-diol), estrone (E1), estradiol (E2), and progesterone (PROG). It also includes eight 11‑oxygenated androgens, comprising the abundant 11β-hydroxyandrostenedione (11OHA4) and its 11β-hydroxylated metabolites: 11-keto-androstenedione (11KA4), 11-hydroxytestosterone (11OHT), 11-keto-testosterone (11KT), 11-hydroxydihydrotestosterone (11OHDHT), 11-keto-dihydrotestosterone (11KDHT), 11β-hydroxyandrosterone (11OHAST), and 11-keto-androsterone (11KAST). It directly quantifies PROG and A4 whereas other steroids are derivatized to enhance sensitivity. Rigorous validation and cross-validation against established assays ensure its reliability and accuracy in steroid quantification. The assay exhibits high sensitivity, achieving a lower limit of quantification as low as 3.7 pM, enabling reliable detection at minimal concentrations. These capabilities are crucial for accurate quantification in hormone-related clinical settings. Clinical applicability is demonstrated using plasma and serum specimens of men with prostate cancer undergoing hormone therapy, underscoring the assay's relevance in contexts of low circulating steroid concentrations. The high sensitivity of this assay warrants its utility in diverse clinical contexts where precise quantification is critical.