Measurement of serum steroid profiles by HPLC-tandem mass spectrometry

Accurate investigations of adrenal hormone levels play a vital role in pediatric endocrinology for the detection of steroid-related disorders. In this study, we developed and validated a simultaneous assay for eight serum steroids, i.e., DHEA, androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, DHEA-sulfate, pregnenolone-sulfate and cholesterol-sulfate, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Serum samples were prepared by liquid-liquid extraction with methyl t-butyl ether. Quantitation by LC-MS/MS was performed in multiple reaction monitoring mode with an electrospray ionization source. The run time was 10 min. Analytical performance was evaluated, including imprecision, linearity, ion suppression, carry over and detection capabilities. Serum specimens from 59 children and 120 adults were analyzed to compare the distribution of steroid levels between the groups. All hormones were effectively extracted and separated using our method. The method was essentially free from potential interference and ion suppression. Within-run and between-run imprecision values were < 20%. The lower limits of quantification varied from 0.025 to 10 ng/mL. The results were generally good and correlated with those obtained using immunoassay techniques. We developed the HPLC-MS/MS method for the simultaneous measurement of free and sulfated steroid hormones. The performance of the developed method was generally acceptable. Thus, this method may provide a novel approach to steroid profiling in children of age before adrenarche.