Multi-class UHPLC-MS/MS method for the simultaneous determination of 32 endocrine-disrupting chemicals in the urine of pregnant women.

Endocrine-disrupting chemicals (EDCs) comprise structurally diverse contaminants that are frequently detected in human biological matrices and may interfere with endocrine homeostasis even at low exposure levels. Developing practical multi-residue methods for biomonitoring remains challenging because EDC classes differ markedly in polarity, ionization behavior, and urinary matrix susceptibility. In this study, we developed and validated an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of 32 target EDCs in urine from pregnant women, including 14 bisphenols, 8 per- and polyfluoroalkyl substances (PFAS), 5 organophosphate ester metabolites, and 5 preservatives. Sample preparation involved enzymatic hydrolysis followed by liquid-liquid extraction with ethyl acetate. The method was evaluated using matrix-matched calibration in artificial urine and isotope-labelled internal standards where available. Most analytes showed acceptable linearity, accuracy, precision, matrix effect, extraction efficiency, and stability according to commonly used bioanalytical validation criteria. Limits of quantification ranged from 0.001 to 0.4 ng/mL. The method was further applied to 188 urine samples from pregnant women, demonstrating its applicability for routine multi-class EDC biomonitoring. Although sensitivity for several phenolic analytes and extraction efficiency for selected compounds require further improvement, the proposed method provides a practical platform for high-throughput assessment of mixed EDC exposure in epidemiological studies.