Background
Reference intervals covering the whole life span for all the metabolites in the steroid hormone biosynthesis quantified by sensitive and robust analytical methods are sparse or not existing.

Objective
To develop a state-of-the-art LC-MS/MS method for simultaneous quantification of multiple steroid metabolites and to establish detailed sex- and age-specific reference intervals for 16 steroid metabolites.

Materials and method
An isotope diluted LC-MS/MS method was developed for simultaneous quantitation of 16 steroid hormones. Serum samples from cross-sectional cohorts of healthy infants, children, adolescents, and adults aged 0.17 months to 77 years (n = 2458) were analysed.

Results
With this novel, specific, and sensitive LC-MS/MS method, it was possible to quantify progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dihydrotestosterone, 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol, and cortisone in ≥90 % of the samples, while estrone sulfate, aldosterone and dehydroepiandrosterone were quantified in 77 %, 75 % and 60 % of the samples, respectively. 21-deoxycortisol was only detectable in 2.5 % of samples from healthy subjects. Sex- and age-dependent fluctuations observed in minipuberty, puberty and adulthood including the menopausal transition were modelled. This enabled us to establish valid reference intervals from birth to late adult life for both males and females.

Conclusion
Detailed sex- and age-specific reference intervals of multiple, simultaneously quantified steroid metabolites by a novel and specific LC-MS/MS method provides a valuable tool for clinical practice and for future research.