Endocrine Abstracts

JOINT655

Steroids are crucial for numerous biological processes, and their dysregulation is linked to various endocrine disorders from hypertension to adrenocortical carcinoma. Accurate steroid profiling is essential for understanding these conditions. Here we present a validated method (EMA/FDA criteria) for urinary steroid profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) which quantifies 29 steroids, including steroid precursors, mineralocorticoids, glucocorticoids, and androgens. Steroids were extracted from urine via C₁₈ solid-phase extraction after deconjugation from their sulfate and glucuronide esters. Quantification was performed using a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Chromatographic separation of 27 steroids was achieved in 16 minutes using a Waters HSS T3 column, with a second injection onto a BEH C₁₈ column to separate and quantify THF and 5αTHF in 4 minutes. The lower limits of quantification ranged from 2 to 20 ng/ml, with accuracy (bias) between 12.6% and 19.9% and precision (%CV) from 4.0% to 18.6%. Imprecision assessed at three concentrations was within acceptable limits for all steroids, ranging from 3.4% to 14.1%. Accuracy (bias) ranged from −14.9% to 14.9%, (excluding 5PD). Recovery was 76–103%, with matrix effects within the ideal range (<15%) for 15 steroids and the acceptable range (<20%) for the rest. Inter- and intra-assay imprecision (%CV) ranged from 0.8% to 14.9%. In a proof or principle cohort of 40 healthy volunteers, all steroids were detectable, with DHEA, α-cortol, THAldo, 5PD, and PTONE quantifiable in 86%, 84%, 72%, 43%, and 38% of samples, respectively. Steroid excretion was higher during the day and in men compared to women. This LC-MS/MS method enables high-throughput, comprehensive multi-steroid quantification across multiple steroid classes, offering a valuable tool for clinical research and diagnostic applications. This method will be implemented in the multi-centre European clinical research study, HT-Advance, where it will be combined with other technologies to rapidly measure urinary steroid excretion, evaluate the diagnostic performance of MOMICS (multi-omics) technologies in identifying endocrine causes of hypertension, and predict treatment responses in patients with newly diagnosed hypertension.