Determination of estrone sulfate, testosterone, androstenedione, DHEAS, cortisol, cortisone, and 17α-hydroxyprogesterone by LC-MS/MS in children and adolescents

Abstract

Quantitation of endogenous steroids and their precursors is essential for diagnosis of a wide range of endocrine disorders. Usually, these analyses have been carried out using immunoassays. However, immunoassays often overestimate concentrations due to assay interference by other endogenous steroids, especially for low concentrations. Mass spectrometry based methods offer superior specificity, accuracy, and sensitivity. We therefore present a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method with automated sample preparation for determination of 17α-hydroxyprogesterone (17OHP), cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A₄), testosterone (T), and estrone sulfate (E₁S). Samples were prepared using protein precipitation and 96-well filter plates, fully automated in a pipetting robot and analyzed by LC-MS/MS. Serum samples from 187 healthy children and adolescents aged 5-18 years were used to study hormone changes in relation to sex and pubertal stage. Lower limit of quantification for 17OHP was 0.7 nmol/L, for cortisol 11 nmol/L, for cortisone 2 nmol/L, for DHEAS 0.1 µmol/L, and for A₄, T, and E₁S, 0.2 nmol/L. This study showed a general increase in 17OHP, DHEAS, A₄, T and E₁S in both genders during puberty. In boys, A₄ and T increased significantly throughout pubertal development. Girls had significantly higher A₄ and E₁S concentrations, while boys had higher T concentrations. No sex- or puberty-specific differences were seen in cortisol or cortisone concentrations. To the best of our knowledge, this is the first presentation of changes in serum E₁S concentrations during pubertal development in healthy children.

Keywords:

• Androgens
• children
• cortisol/cortisone ratio
• estrone sulfate
• mass spectrometry
• puberty

Acknowledgements

The authors thank the staff at the Göteborg Pediatric Growth Research Center laboratory for sample handling and Monika Eriksson for excellent technical assistance.

Author contributions

All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement (427731), the Swedish Research Council (521-2013-3013), and the IngaBritt and Arne Lundberg Research Foundation (2015-103). The funding organizations played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.