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The influence of sex, age, pubertal development and oral contraceptives on dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), testosterone (T), calculated free testosterone (fT), free androgen index (FAI) and selected ratios in 1798 serum samples from healthy children, adolescents and young adults was evaluated. Samples were analyzed by Turboflow-LC–MS/MS. Sex hormone-binding globulin was analyzed by immunoassay. All steroid metabolite concentrations were positively associated with age and pubertal development in both sexes and generally higher in males than in females except for Adione. The pubertal rise in T in males was more pronounced compared to females, reflecting contribution from the testes. Ratios between steroid metabolites varied and depended on sex and age. All ratios were lower during infancy compared to later in life. Use of oral contraceptives significantly lowered serum concentrations of all steroid metabolites, fT, FAI, the 17-OHP/Adione, the Adione/T and the DHEA/Adione ratios, but not the DHEA/DHEAS ratio. We provide reference ranges for DHEA, DHEAS, 17-OHP, Adione, T, fT, FAI and selected ratios in relation to sex, age and pubertal development. Use of oral contraceptives strongly influences adrenal steroidogenesis and should be considered when diagnosing and monitoring treatment of patients with disorders of sex development. [ABSTRACT FROM AUTHOR]

Abstract: Diagnosis and management of infants and children with sex steroid disorders require fast and simultaneous assessment of several sex steroid metabolites in serum at low concentrations and on small sample volumes. Therefore, we developed a sensitive and selective TurboFlow-LC–MS/MS method for quantification of DHEA, DHEAS, 17α-hydroxyprogesterone, Δ4-androstenedione and testosterone in serum from pre-pubertal children. Run time was 10.75min. Limits of quantification were as follows: DHEA, 0.88nM; DHEAS, 48nM; 17α-hydroxyprogesterone, 0.19nM; Δ4-androstenedione, 0.18nM and testosterone, 0.10nM. Intra-day relative standard deviation ranged from 4.6 to 13.8% and inter-day relative standard deviation ranged from 5.7 to 15.7%. Steroid concentrations in 38 serum samples from pre-pubertal children were compared with results obtained by immunoassays for DHEAS, Δ4-androstenedione and testosterone. DHEAS gave overall similar results but with several outliers, while levels of Δ4-androstenedione were found to be much lower when analysed by LC–MS/MS. Testosterone was not detected in any of the samples analysed using a sensitive immunoassay, while 30 of 38 samples were quantifiable using the current LC–MS/MS method. The presented method is suitable in a clinical setting for simultaneous quantification of five steroids important for management of children with disorders of sex development and steroid biosynthesis defects. [Copyright &y& Elsevier]

The adrenal and gonadal androgens, testosterone, androstenedione and dehydroepiandrosterone (DHEA) play an important role in sexual development as well as in other processes. We developed a method for simultaneous quantitative analysis of serum and plasma testosterone, androstenedione and DHEA levels using Isotope-Dilution Liquid-Chromatography Tandem Mass Spectrometry (ID-LC–MS/MS). Samples underwent liquid–liquid extraction and were analyzed on an Acquity 2D-UPLC-System and a Xevo TQ-S tandem mass spectrometer (Waters). The intra-assay and inter-assay coefficients of variation were < 4.0%, < 6.3% and < 7.0% and < 6.0%, < 8.1% and < 7.7% for testosterone, androstenedione and DHEA, respectively. Inter-assay CVs at the lower limit were 10.6%, 16.9% and 9.0% for testosterone (0.10 nmol/L), androstenedione (0.10 nmol/L) and DHEA (1.0 nmol/L), respectively. Recoveries of spiked analytes were 93–107%. The present testosterone method compared well (y = 1.00x − 0.04; r = 0.998) to a published ID-LC–MS/MS method for testosterone in our lab. The latter method being concordant with a published reference method (Bui et al., 2013). The present method compared well to a published ID-LC–MS/MS method (Kushnir et al., 2010) (y = 1.06x − 0.06; r = 0.996 for testosterone; y = 1.04x − 0.04; r = 0.995 for androstenedione and y = 1.03x + 0.01; r = 0.991 for DHEA). In conclusion, we developed a sensitive and accurate ID-LC–MS/MS method to simultaneously measure serum testosterone, androstenedione and DHEA in serum and plasma. [ABSTRACT FROM AUTHOR]