Determination of reference intervals for urinary steroid profiling using a newly validated GC-MS/MS method

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Urinary steroid profiling (USP) is a powerful diagnostic tool to asses disorders of steroidogenesis. Pre-analytical factors such as age, sex and use of oral contraceptive pills (OCP) may affect steroid hormone synthesis and metabolism. In general, USP reference intervals are not adjusted for these variables. In this study we aimed to establish such reference intervals using a newly-developed and validated gas chromatography with tandem mass spectrometry detection method (GC-MS/MS).


Two hundred and forty healthy subjects aged 20-79 years, stratified into six consecutive decade groups each containing 20 males and 20 females, were included. None of the subjects used medications. In addition, 40 women aged 20-39 years using OCP were selected. A GC-MS/MS assay, using hydrolysis, solid phase extraction and double derivatization, was extensively validated and applied for determining USP reference intervals.


Androgen metabolite excretion declined with age in both men and women. Cortisol metabolite excretion remained constant during life in both sexes but increased in women 70-79 years of age. Progesterone metabolite excretion peaked in 30-39-year-old women and declined afterwards. Women using OCP had lower excretions of androgen metabolites, progesterone metabolites and cortisol metabolites. Method validation results met prerequisites and revealed the robustness of the GC-MS/MS method.


We developed a new GC-MS/MS method for USP which is applicable for high throughput analysis. Widely applicable age and sex specific reference intervals for 33 metabolites and their diagnostic ratios have been defined. In addition to age and gender, USP reference intervals should be adjusted for OCP use.

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