Measurement of serum testosterone using high-performance liquid chromatography/tandem mass spectrometry

Low levels of serum testosterone typically found in women and children cannot be reliably measured by immunoassay. We developed a simple and sensitive method using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Sample preparation involved protein precipitation of serum (1.0 mL) with acetonitrile containing the internal standard (testosterone-d3) followed by liquid extraction with methylene chloride. The chromatographic cycle per specimen was 10 min. The performance was evaluated according to the CLSI EP10-A2 protocol. Within- and between-run imprecision was 20.9%, 2.29% and 1.80%, and 1.81%, 3.58% and 2.97% at mean concentrations of 0.17, 14.1 and 28.8 nM, respectively, with no apparent carryover. The method was linear from 0.21 to 53.1 nM and the analytical recovery was 100.5-106.2% across the concentrations tested. There was no interference observed from other steroids that were tested. Correlation using de-identified patient specimens with a commercial HPLC-MS/MS method showed a slope of 0.991, an intercept of -0.017 and a correlation coefficient (R(2)) of 0.998 by linear regression over concentrations ranging from 0.21 to 16.7 nM. In conclusion, we report here an HPLC-MS/MS method suitable for clinical measurement of serum testosterone.