A sensitive HPLC-DMS/MS/MS method for multiplex analysis of androgens in human serum without derivatization and its application to PCOS patients

Abstract Both classical androgens and 11-oxygenated androgens play important roles in polycystic ovarian syndrome (PCOS). Therefore, high-quality measurements of androgens are very important. In the present study, a highly sensitive and specific method was developed and validated for the simultaneous determination of three classical androgens and five 11-oxygenated androgens in human serum, using a high- performance liquid chromatography-differential mobility spectrometry tandem mass spectrometry (HPLC-DMS/MS/MS). Serum samples were extracted with the mixture of ethyl acetate/tert-butyl methyl ether (1/1, v/v) prior to analysis with the HPLC-DMS/MS/MS system. Stable isotopes were used as the internal standards. Separation was performed on a Poroshell SB C18 column (150 × 2.1 mm, 2.7 µm), with a differential mobility spectrometry (DMS) component, which was used to enhance the resolution. The gradient mobile phase consisted of acetonitrile and ammonium formate buffer with 0.1% formic acid in both solvents. The sensitivity of the majority of the androgens was improved following addition of the DMS component. Under the optimal conditions, the trace amount of the target androgens in the serum was quantified accurately. The lower limit of quantification of the different analytes ranged from 0.05 to 0.2 ng/mL. The method was validated prior to its application to the assay of the clinical samples.