B-193 Establishing a robust liquid chromatography-tandem mass spectrometry method for urinary steroid profiling with efficient enzymatic pretreatment

Background Abnormal steroid secretion status has been implicated in a variety of adrenal diseases, such as primary aldosteronism (PA), and adrenocortical carcinoma. However, existing methodological studies to quantify urinary steroids have limitations such as few measurable hormones, complex and time-consuming pre-processing, etc. Therefore, the study aims to establish a robust and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for multi-steroid profiling in human 24-hour urine, promoting individualized diagnosis. Methods IMCS RT transgenic ß-glucuronidase (activity>200kU/mL) was used to hydrolyze urine samples, with the performance under different hydrolysis durations (0, 0.25, 0.5, 1, 2, 4 hours) evaluated. A 96-well µHLB solid-phase extraction plate was used to extract steroids which were then detected using a Waters Acquity I-Class UPLC system in tandem with a Waters TQ-S triple quadrupole MS/MS system. The performance of the LC-MS/MS method was validated according to the Clinical & Laboratory Standards Institute guidelines. The distribution of 24-hour urinary steroid hormones in sixteen patients with PA and seventeen healthy participants was compared. Results Steroids in 40µL 24-hour urine could be effectively hydrolyzed with 5µL IMCS RT ß-glucuronidase at room temperature for approximately 30 minutes. Positive electrospray ionization using multiple reaction modes was performed for most hormones, except for estrone, estradiol, estriol, aldosterone, and tetrahydroaldosterone. Thirty-five steroids were effectively chromatographic separated using an Acuqity UPLC® HSS C18 column and analyzed in one injection within 25 minutes. The linearity range was 0.012-10ng/mL for 4-androsten-3,11,17-trione, 0.12-100ng/mL for estrone, estradiol, estradiol, 18-oxocortisol, 11-deoxycorticosterone, 18-hydro corticosterone and aldosterone, 1.2-1000ng/mL for androstenedione, dehydroepiandrosterone, 11-oxo etiocholanolone, 6ß-hydroxy cortisol and tetrahydro-11-deoxycorticosterone, 0.024-20ng/mL for 11-ketotestosterone, 0.24-200ng/mL for 18-hydroxycortisol and 11-deoxycortisol, 2.4-2000ng/mL for androsterone and ß-cortolone; 0.006-5ng/mL for 17a-hydroxyprogesterone, 0.06-50ng/mL for dihydrotestosterone, dexamethasone, testosterone and 11-hydroxy progesterone, 0.6-500ng/mL for progesterone, cortisone, cortisol and tetrahydroaldosterone, 6-5000ng/mL for 11ß-hydroxy etiocholanolone, tetrahydrocortisol, 5ß-Pregnane-3a,20a-diol, a-cortolone, urocortisone, allo-3a-tetrahydrocortisol, 11ß-hydroxy androsterone, and 5ß-androstan-3a-ol-17-one. The recovery rate was between 85% and 115%. No obvious matrix effect was observed with internal standard correction. Acceptable intra-assay and inter-assay precision were achieved for all analytes with the total coefficients of variation being <20% at low concentrations, and <15% at medium and high concentrations. No carryover was found. Patients with PA showed significantly different steroid secretion conditions when compared with healthy participants, such as higher urinary median values of aldosterone (16.4 vs. 4.0µg/24h), tertrahydroaldosterone (91.9 vs. 14.4µg/24h), 18-oxocortisol (2.7 vs. 1.3µg/24h), 18-hydroxycortisol (143.6 vs 105.8µg/24h), etc., but lower 11ß-hydroxy etiocholanolone (69.6 vs. 191.7µg/24h) (all p<0.05). Conclusion This LC-MS/MS, combined with a shorter hydrolysis time, enables simultaneous quantification of multiple steroids in a single run, allowing for fast, robust, and accurate applications in disease diagnosis.