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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Endocrinology

Background:

• Glucocorticoids are key biomarkers for stress response and disease, regulating metabolism and immunity.
• Current analytical methods often require large sample volumes, limiting large-scale studies.
• Downscaling sample requirements is crucial for advancing research in these areas.

Purpose of the Study:

• To develop and validate a downscaled liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for analyzing urinary cortisol and cortisone.
• To reduce the required urine sample volume for glucocorticoid analysis while maintaining accuracy and reliability.

Main Methods:

• Modified an existing LC-MS/MS method, decreasing urine sample volume from 100 μL to 20 μL.
• Incorporated cortisone-d8 as an internal standard and adjusted calibration standards (1/5 dilution).
• Validated linearity, lower limit of quantitation (LLOQ), matrix effects, recovery, accuracy, and reproducibility.

Main Results:

• Achieved good linearity (2-200 ng/mL) with an LLOQ below 2 ng/mL.
• Observed acceptable coefficients of variation (<15%) for matrix factors.
• The downscaled method demonstrated good recovery, accuracy, reproducibility, and agreement with the original method, with some matrix effect variations.

Conclusions:

• The downscaled LC-MS/MS method effectively analyzes urinary cortisol and cortisone using significantly reduced sample volumes.
• This optimized method facilitates larger cohort studies and accelerates research on stress response and related diseases.
• The method shows potential for advancing the study of glucocorticoids in various physiological and pathological conditions.