Targeted LC-MS/MS analysis of steroid glucuronides in human urine

Area of Science:

• Endocrinology and Metabolism
• Analytical Chemistry
• Biochemistry

Background:

• Steroid hormone catabolism involves conjugation with glucuronic acid, a major metabolic pathway.
• Growing interest exists in understanding the biological functions of steroid glucuronides.
• Accurate quantification of urinary steroid glucuronides is crucial for metabolic studies.

Purpose of the Study:

• To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of 15 urinary steroid hormone glucuronides.
• To apply the validated method to analyze steroid hormone metabolites in urine samples from children and adolescents.
• To investigate age-related changes in the excretion patterns of steroid hormone conjugates.

Main Methods:

• Development and validation of an LC-MS/MS method for quantifying 15 specific urinary steroid hormone glucuronides.
• Sample preparation involving protein precipitation and solid-phase extraction.
• Application of the method to 67 urine samples from children and adolescents, with comparison to GC-MS data.

Main Results:

• The LC-MS/MS method demonstrated excellent linearity (R²≥0.99), recovery (89.6–113.8%), and quantification limits (1.9–21.4 nmol/L).
• Accuracy and precision were consistently below 15% for all quality controls.
• Analysis revealed age-dependent changes in steroid hormone conjugation, with glucuronides dominating for androgens and sex hormones, while sulfates were prominent for dehydroepiandrosterone (DHEA) and cortisol in newborns.

Conclusions:

• The validated LC-MS/MS method provides a reliable tool for simultaneous quantification of multiple urinary steroid hormone glucuronides.
• The study highlights significant age-related shifts in steroid hormone metabolism and conjugation patterns throughout childhood and adolescence.
• Findings underscore the importance of considering different conjugation forms (glucuronides and sulfates) for a comprehensive understanding of steroid hormone homeostasis.