High-sensitivity quantification of serum androstenedione, testosterone, dihydrotestosterone, estrone and estradiol by gas chromatography-tandem mass spectrometry with sex- and puberty-specific reference intervals

Abstract

Background: Androgen and estrogen determinations serve as important diagnostic markers in a variety of clinical conditions. However, one challenge is to enhance assay sensitivity for determination in the lowest range, such as in prepubertal children. We here present a recently developed gas chromatography-tandem mass spectrometry (GC-MS/MS) method for determination of androstenedione (A₄), dihydrotestosterone (DHT), testosterone (T), estrone (E₁), and estradiol (E₂) in children, which we have compared with the sensitive radioimmunoassays; E₂ extraction-RIA and T-RIA.

Methods: Steroids were extracted in ethyl acetate n-hexane solution from serum spiked with isotopically labeled internal standard and derivatized sequentially with pentafluorobenzyl bromide, pentafluorobenzyl hydroxylamine and pentafluoropropionic acid anhydride and analyzed by GC-MS/MS using a triple quadrupole mass spectrometer operated in negative chemical ionization mode. Leftover routine samples (n = 414) were used to evaluate the concordance between GC-MS/MS and RIAs and the validity of GC-MS/MS for pediatrics; of these samples, 101 were from seemingly healthy children. Pubertal stage was recorded for reference interval evaluation.

Results: Lower limit of detection for A₄, T, DHT, E₁, and E₂ were 0.1 nmol/L, 0.1 nmol/L, 27 pmol/L, 9 pmol/L, and 2 pmol/L, respectively. Good agreement was found between GC-MS/MS and T-RIA (r = 0.98) as well as between GC-MS/MS and E₂ extraction-RIA (r = 0.98, for E₂ concentrations above 14 pmol/L). In boys, T and DHT increased significantly from prepuberty throughout pubertal development, and in girls the same increase was observed for E₁ and E₂. The greatest increase in A₄ for both genders, as well as E₁ and E₂ in boys and T and DHT in girls, occurred in mid to late puberty.

Conclusions: We report the development of a GC-MS/MS method sensitive enough to accurately determine serum levels of androgens and estrogens in children.

Keywords: Androgens; Children; Estrogens; Mass spectrometry; Reference intervals.