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

Publication date: Available online 9 June 2018
Source:The Journal of Steroid Biochemistry and Molecular Biology
Author(s): Carina Ankarberg-Lindgren, Jovanna Dahlgren, Mats X. Andersson
BackgroundAndrogen 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.MethodsSteroids 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.ResultsLower 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.ConclusionsWe report the development of a GC-MS/MS method sensitive enough to accurately determine serum levels of androgens and estrogens in children.



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