Rapid analysis in small-volume urine: Optimizing LC-MS/MS for free glucocorticoids

Glucocorticoids, regulators of energy metabolism and immune responses, are known biomarkers of stress response and disease. Downscaling the required sample volumes facilitates large-scale studies and accelerates the generation of valid results. To meet this expectation, we modified our LC-MS/MS method using 100 μL of urine to that using 20 μL, following the same steps as in the original protocol with the addition of cortisone-d8 as an internal standard. Calibration was performed as in the original protocol but using 1/5 diluted standards, resulting in a good linearity of 2-200 ng/mL in the log-log plot, indicating a lower limit of quantitation (LLOQ) of <2 ng/mL from the residue. The coefficients of variation of the matrix factors of the 15 urine samples and water were within 15 % for 10 and 300 ng/mL cortisol and cortisone. The matrix factor of the downscaled method was larger than that of the original method, especially for urine extracts with high specific gravity (up to 2.1-fold), indicating the advantage of the downscaled method; although the theoretical LLOQ is 5-fold with the 1/5 downscaling, the effect on the practical LLOQ may be reduced by the suppressed matrix effect. The current method showed good recovery, accuracy, reproducibility, and agreement with the original method (regression coefficient close to 1.0). After sample freezing, cortisol and cortisone were detected at higher and lower levels, respectively, within 25 %. The downscaled method of urine-free cortisol and cortisone may contribute to the advancement of related research.