Comprehensive Quantitative Urinary Steroid Profiling of 29 Steroids Using Liquid Chromatography-Tandem Mass...

Steroids are critical for numerous physiological processes; disruption in their metabolism is associated with numerous endocrine disorders. Steroid quantification is essential to improve the understanding and diagnosis of these pathologies. Historically, urinary steroid profiling has been performed using low-throughput gas chromatography mass spectrometry (GC-MS), providing holistic coverage of steroid classes with low cross-reactivity. Here, we translate our previous GC-MS urinary steroid profile to a liquid chromatography tandem-MS (LC-MS/MS) platform, offering a validated, comprehensive overview of steroid metabolism with comparatively low sample preparation times and increased throughput. Urinary steroids were enzymatically hydrolysed and extracted via C₁₈ solid-phase extraction. Quantification was conducted using a triple quadrupole mass spectrometer. Chromatographic separation of 27 analytes was completed in 16 min using a C₁₈-T3 column. Due to chromatographic co-elution of tetrahydrocortisol and 5α-tetrahydrocortisol, a second injection was required on a BEH-C₁₈ column for their separation. Lower limit of quantification (LLOQ) ranged from 2 to 20 ng/mL, witha ccuracy (bias) ranging from -18.7% to 19.9%, and precision (percentage coefficient of variation [%CV]) ranging from 4.0% to 18.6%. Matrix effects remained within the ideal range <±15% for all steroids. Recovery ranged from 76% to 103%, and intra- and inter-assay imprecision (CV) ranged from 0.8% to 14.9%. In 40 healthy volunteers, most analytes were detected above the LLOQ in over 95% of samples, although tetrahydro-aldosterone (85%), 5-pregenediol (68%), and pregnanetriol-one (59%) demonstrated lower quantification rates. Diurnal and sex-based variations were observed, with excretion levels significantly higher during daytime and in males. This robust, high-throughput LC-MS/MS method facilitates the simultaneous quantification of multiple steroid classes, enhancing its utility for clinical and research applications in endocrine science.