Simultaneous determination of androstenedione and testosterone in human serum by liquid chromatography-tandem mass spectrometry

Background We aimed to develop a sensitive assay to quantitate serum concentrations of both androstenedione and testosterone within the female range simultaneously, using liquid chromatography-tandem mass spectrometry (LC-MS/ MS) for use in the routine clinical laboratory and to compare this method with immunoassay. Method Samples (200 mu L) were prepared by liquid-liquid extraction using (1 mL) methyl-tert-butyl-ether. Deuterated androstenedione and testosterone were used as internal standards. Results The standard curve was linear to 50 nmol/L, the lower limit of quantitation was 0.25 nmol/L, and intra- and inter-assay coefficients of variation were < 10% for both androgens over the range 0.3-35 nmol/L. There was a poor relationship between the LC-MS/MS and the radioimmunoassay methods for androstenedione with the LC-MS/MS generally giving lower results. For testosterone, the LC-MS/MS and immunoassay methods compared well at all concentrations. However, when female samples only were examined, the agreement deteriorated. Conclusions We have developed a sensitive and precise LC-MS/MS method, which gives more accurate results for all androstenedione measurements and low testosterone concentrations than immunoassay.