Establishing and Verifying a Robust Liquid Chromatography-Tandem Mass Spectrometry Method to Simultaneously Measure Seven Androgens Present in Plasma Samples

Abstract

Objectives: To develop a robust liquid chromatography-tandem mass spectrometry (LC–MS/MS) method to simultaneously measure seven human plasma androgens, namely testosterone (T), dihydrotestosterone (DHT), androstenedione (A4), dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), 11-ketotestosterone (11-KetoT), and 11β-hydroxytestosterone (11β-OHT). Design and Methods: Plasma was extracted via a solid phase extraction method, and the analytical performance of the assay was validated according to the Clinical & Laboratory Standards Institute guidelines. Overall, 73 apparently healthy volunteers were recruited to evaluate the distribution of these seven androgens; their levels in 25 females with acne and 33 obese females were also evaluated. Results: The developed method exhibited a good precision, with the total coefficient variations (CV) and the intra-assay CVs being within 10%. Furthermore, the recoveries of T, DHT, A4, DHEA, DHEAS, 11-KetoT, and 11β-OHT were 90.3–105.8, 88.7–98.1, 92.4–102.5, 90.5–106.7, 87.6–99.9, 93.3–105.3, and 90.2–104.4%, respectively, and no significant matrix effect was observed after internal standard correction (<20%). Moreover, the limits of quantification were 0.01, 0.01, 0.01, 0.10, 5.00, 0.02, and 0.02 ng/mL for T, DHT, A4, DHEA, DHEAS, 11-KetoT, and 11β-OHT, respectively, which are adequate for their accurate measurement in human plasma samples. It was also determined that patients diagnosed with acne had significantly higher levels of DHT, A4, and DHEAS, while those suffering from obesity had significantly higher levels of T and A4 but lower levels of DHT. Conclusions: A robust LC-MS/MS method for the simultaneous determination of seven androgens in plasma samples was successfully established and validated, which plays important roles in clinical application.