A-097 Exploring a Simplified Urine Collection Method for Estimating 24-Hour Steroid Results: A Pilot Study

Abstract Background The 24-hour urine collection method has traditionally been used in clinical settings to measure various biomarkers. However, this method is time-consuming and prone to errors, leading to variability in results. This study evaluates the effectiveness of collecting urine at four specific time points throughout the day as an alternative to the traditional 24-hour collection for glucocorticoid, androgen, estrogen, and other steroid hormones. Methods Six donors (3 male, 3 female, including post- and premenopausal, collected during the late luteal phase) collected urine voids over a 24-hour period. Each void volume was recorded. A small aliquot from each void was analyzed individually, while the rest of the voids were pooled and analyzed as a 24-hour sample. Urine samples were hydrolyzed using beta-glucuronidase and estrogens derivatized as described previously.1 All steroid and creatinine measurements were performed via LC-MS/MS. Steroid hormone concentrations from four distinct time points (first morning, ∼2 hours post-waking, evening, and late night) were compared against a full 24-hour collection as "creatinine normalized" and "24-hour predicted" results (based on 20 mg creatinine production per kg body weight for males, 15.5 mg for females).2 Additionally, the 4-point collection was compared against first morning and all collection average to estimate the 24-hour collection. Slopes and correlation coefficients (R²) were calculated. Results Our findings show that the 4-point collection highly correlated with the full 24-hour collection. Observed 4-point results were within 92-98% of the 24-hour data for many steroid hormones. “Creatinine normalized” cortisol (F) showed a slope close to 1, but an R² of 0.75. Cortisol metabolites (5A-THF and THF) 4-point “24-hour predicted” results matched measured 24-hour results (slopes within 1.1, R² = 0.93). Estradiol (E2), testosterone (T), EPI-T, and 5A-DHT matched 24-hour results both as “creatinine normalized” and “24-hour predicted” (slopes ˜ 1, R² = 0.9). Major urinary progesterone (P4) metabolites 5A-PD and 4B-PD were slightly underestimated via the 4-point collection (lowest slope of 0.85), but showed excellent correlation for “creatinine normalized” results (lowest R² of 0.98). Furthermore, this method exhibited reduced variability, demonstrating improvement in consistency and considerable reduction in collection time. Conclusion These results suggest that collecting urine at four specific time points throughout the day is as effective and reliable as a full 24-hour collection for many steroid hormones, while offering a more practical approach. This method not only reduces patient burden but also maintains analytical reliability, making it an ideal solution for clinical and research settings. This work opens the door to further investigation in simplifying urine collection strategies for steroid hormones and other urinary analytes where 24-hour collection is not possible.