Zhimin Zhou, Yang Wang, Zengyue Li, Yanying Li, Ruhan Gong, Jinyu Yang
Steroid hormones serve as critical biomarkers reflecting human physiological endocrine status, once excreted and accumulated in aquatic environments, these compounds transform into typical endocrine-disrupting chemicals (EDCs) that threaten human health and aquatic ecosystems even at trace concentrations. To investigate the presence and risk of steroids in the human - environment cycle, this study developed and validated a robust, sensitive, and reproducible method based on solid-phase extraction coupled with ultra-high liquid chromatography tandem-mass spectrometry for the quantification of 16 steroids across multiple matrices - plasma, urine and wastewater. The method was fully validated according to EMA guidelines, showing excellent linearity (R 2 > 0.99 for all analytes), recoveries ranging from 84.79% to 111.57% across all matrices, and precision with intra-day RSDs of 0.55–11.54% and inter-day RSDs of 1.69–10.37%. Matrix effects were effectively corrected using isotopically labeled standards, ranging from 85.61% to 113.04%. The total analysis time was 7 minutes per sample. The detection rate of 87.5%, 100%, and 100% of target analytes in real plasma, urine, and wastewater matrices, respectively, demonstrated the feasibility of the method for multi-matrix applications. Substantial concentration differences were observed across matrices, with DHEAS being the most abundant analyte in all sample types. This multi-matrix approach enables comprehensive monitoring of steroid exposure from individual to population levels, supporting environmental health assessments and wastewater–based epidemiology analysis.