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Background Liquid-chromatography tandem mass spectrometry (LC–MS/MS) has become the method of choice in steroid hormone measurement. However, little information on the mutual agreement of LC–MS/MS methods is available. We compared eight routine unpublished LC–MS/MS methods for the simultaneous measurement of testosterone and androstenedione. Methods Sixty random serum samples from male and female volunteers were analysed in duplicate by eight routine LC–MS/MS methods. We performed Passing–Bablok regression analyses and calculated Pearson's correlation coefficients to assess the agreement of the methods investigated with one published method known to be accurate. Intra-assay CV of each method was calculated from duplicate results, recoveries for each method were calculated from six spiked samples. Furthermore, a CV between the investigated methods was calculated. Results The concentrations ranged from 0.05–1.26 nmol/L, 6.15–24.44 nmol/L and 0.15–4.78 nmol/L for testosterone in females, testosterone in males and androstenedione, respectively. The intra-assay CVs were between 3.7–16.0%, 0.9–5.2% and 1.2–9.5% for testosterone in females, testosterone in males and androstenedione, respectively. The slopes of the regression lines ranged between 0.90–1.25, 0.87–1.24 and 0.94–1.31 for testosterone concentrations in females, all testosterone values and androstenedione, respectively. Inter-method CVs were 24%, 14% and 29% for testosterone for concentrations in females and males and androstenedione, respectively. These compare unfavourably to the variation found earlier in published methods. Conclusion Although most routine LC–MS/MS methods investigated here showed a reasonable agreement, some of the assays showed a high variation. The observed differences in standardization should be taken into account when applying reference values, or should, preferably, be solved. [ABSTRACT FROM AUTHOR]

We report reference ranges based on LC–MS/MS for testosterone (T), free testosterone (FT) and its precursors, i.e. 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS) and androstenedione (Adione), in relation to different health markers and lifestyle factors. The study was based on 304 healthy men aged 30–61 years participating in a population-based cross-sectional study (Health2008). Examination program consisted of a clinical examination, completion of a self-administered questionnaire and blood sampling. Steroid metabolites were measured by a validated and sensitive LC–MS/MS method. Older age-groups were significantly associated with decreased concentrations of DHEA, DHEAS, Adione, and FT, while no significant associations with age were shown for 17-OHP or T. Participants with BMI ≥ 30 kg/m 2 had lower age-related steroid metabolite z-scores compared to participants with BMI < 30 kg/m 2 , i.e. 17-OHP: − 0.51 vs. 0.08 (p < 0.001); DHEA: − 0.27 vs. 0.09 (p = 0.014); Adione: − 0.29 vs. 0.09 (p = 0.012); T: − 0.99 vs. 0.14 (p < 0.001); and FT − 0.55 vs. 0.05 (p < 0.001), respectively. In conclusion, this large study on serum steroid metabolites and concomitant assessment of health markers in healthy men provides age-related reference ranges, and furthermore evaluates the impact of lifestyle factors and metabolic syndrome on androgen metabolite levels. [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]