Abstract: Steroid hormones are key endogenous molecules that maintain physiological homeostasis and are crucial for disease diagnosis and treatment. Abnormal serum concentrations of these hormones are closely associated with various endocrine disorders. Traditional immunoassay methods, due to insufficient specificity, cross-reactivity, and limitations in single-analyte detection, cannot meet the clinical demand for multi-analyte combined diagnostics. Liquid chromatography-tandem mass spectrometry (LC-MS), with its high specificity, high sensitivity, and capability for simultaneous detection of multiple targets, has become the "gold standard" for steroid hormone quantitative analysis. This article systematically reviews the methodological breakthroughs of LC-MS technology in serum steroid detection in recent years, focusing on: (1) innovations in sample pretreatment technologies, including efficient automated strategies such as cold-induced phase separation, online solid-phase extraction (Online-SPE), solid-supported liquid-liquid extraction (SLE), and magnetic solid-phase extraction (MSPE); (2) optimization of chromatography-mass spectrometry analytical methods, achieving isomer separation and femtogram-level detection limits through rational selection of biphenyl columns, ammonium fluoride mobile phase additives, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI); (3) evolution of quantification strategies, from traditional stable isotope-labeled internal standard methods to single-point internal calibration strategies, substantially improving multi-component analysis efficiency; (4) evolution of application scenarios. Despite current challenges including insufficient standardization, matrix effects, difficulties in isomer separation, and high equipment and personnel requirements, LC-MS/MS will undoubtedly play an increasingly valuable role in precision medicine and disease prevention with the development of nanomaterial innovations in pretreatment, intelligent data analysis, and miniaturized mass spectrometry technologies. This review provides technical references for clinical laboratories and scientific research.

Key words: liquid chromatography-mass spectrometry, serum, steroids, methodology