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

Highlights • A GC–MS/MS based method for quantification of sex steroids in children is presented. • Adrostenedione, dihydrotestosterone, testosterone, estrone, estradiol were analyzed. • The GC–MS/MS- based method was sensitive enough for use in pediatric applications. • Reference intervals were established in relation to sex and pubertal maturation. 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 (A 4), dihydrotestosterone (DHT), testosterone (T), estrone (E 1), and estradiol (E 2) in children, which we have compared with the sensitive radioimmunoassays; E 2 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 A 4 , T, DHT, E 1 , and E 2 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 E 2 extraction-RIA (r = 0.98, for E 2 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 E 1 and E 2. The greatest increase in A 4 for both genders, as well as E 1 and E 2 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. [ABSTRACT FROM AUTHOR]