Development of a high-throughput LLE-UPLC-QqQ-MS/MS method and comprehensive profiling of urinary steroid hormones in...

Steroid hormones (SHs) in human urine are critical for understanding physiological processes, but their analysis is challenged by intricate urine matrices and diverse SHs physicochemical properties. Existing methods often lack sensitivity, throughput, or comprehensiveness for simultaneous qualitative and quantitative profiling. Thus, there is a need for a state-of-the-art, ultra-sensitive, high-throughput method to comprehensively and simultaneously characterize urinary SHs.

The improved liquid-liquid extraction (LLE) and ultra-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method, developed by optimizing mass spectrum parameters and pretreatment conditions to address urine matrix complexity and SHs diversity, identified 133 SHs and quantified 103. The improved experimental protocol was as follows: sample volume, 1 mL; extraction solvent, methyl tert-butyl ether/ethyl acetate (MTBE/EtAc, 1/1, v/v); solvent volume, 2 mL; extraction times, 2; pH, 9; enzyme activity, 1700 U/mL; eluent A, water; eluent B, methanol. Post-extraction samples were analyzed using UPLC-QqQ-MS/MS, with quantification of method-validated SHs reliably performed through internal standards (ISs) calibration. Validated metrics included excellent linearity (R² > 0.990, 1-500 ng/mL), the method detection/quantitation limit (MDL, 0.003-0.740 ng/mL; MQL, 0.01-2.465 ng/mL), matrix effects (ME, -0.46 %-46.56 %), recoveries (72.31 %-129.80 %), and precision/stability (RSDs <20 %). Applied to 76 elderly adults, it revealed gender-specific temporal variations, with significant 5-month declines in androgens and glucocorticoids.

This method enables comprehensive, sensitive urinary SHs profiling, offering a powerful tool for in-depth research on elderly hormonal dynamics. Its ability to capture gender and temporal variations enhances understanding of age-related hormonal changes.