Liquid Chromatography–Tandem Mass Spectrometry Assay for Androstenedione, Dehydroepiandrosterone, and Testosterone with Pediatric and Adult Reference Intervals

Abstract

Measurement of serum androgens is important in adult, geriatric, pediatric endocrinology, and oncology patients. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of androstenedione, dehydroepiandrosterone (DHEA), and testosterone in these patients. We spiked 200 muL of serum or plasma with isotope-labeled internal standards and performed extraction with methyl t-butyl ether. We then derivatized the extracts with hydroxylamine and analyzed them by LC-MS/MS using a 2-dimensional chromatographic separation with a 3.5-min analysis time. Total imprecision for each analyte was <11.2%. Limits of quantification were 10, 50, and 10 ng/L for androstenedione, DHEA, and testosterone, respectively. Reference intervals were established for children (age 6 months to 17 years), men, and women. Androstenedione and DHEA concentrations were lowest in 2- to 3-year-old children. Adult concentrations were achieved in girls at Tanner stage 3 and in boys at Tanner stage 4-5. In premenopausal and (postmenopausal) women the median concentrations of androstenedione, DHEA, and testosterone were 810 (360), 3000 (1670), 270 (180) ng/L, respectively. In postmenopausal women, concentrations of testosterone were age independent, whereas androstenedione and DHEA concentrations decreased with age. In men the median concentrations of androstenedione, DHEA, and testosterone were 440, 2000, and 3700 ng/L, respectively. In men older than 40 years, median concentrations decreased at rates of 5%, 10%, and 20% per decade for androstenedione, DHEA, and testosterone, respectively. This LC-MS/MS method has the required lower limit of quantification and specificity for analysis of endogenous concentrations of androgens in all groups studied. Reference intervals were established for healthy children and adults.