Ultra‐performance liquid chromatography/tandem mass spectrometric determination of testosterone and its metabolites in in vitro samples

This paper describes the development and partial validation of a fast, sensitive and specific ultra‐performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of testosterone (T) and its four metabolites, 6 β ‐OH‐T, 16 α ‐OH‐T, 16 β ‐OH‐T and 2 α ‐OH‐T, in in vitro samples. The analytical method involves direct dilution of samples with acetonitrile containing an internal standard, followed by separation of testosterone and the four metabolites on an Acquity UPLC™ C 18 column and detected by selected reaction monitoring (SRM) in positive ionization mode using turbo ionspray ionization. The parent compound and its metabolites investigated were well separated (Rs >1.5) with a run time of 4 min under a gradient condition. The method was partially validated. The linear concentration range was 0.01 to 5 µM for all the compounds of interest. Inter‐assay mean bias and relative standard deviation (RSD) were in the range of −12% to 8% and 4.1% to 8.5%, respectively. Intra‐assay mean bias and RSD were in the range of −8.0% to 5.2% and 3.4% to 9.6%, respectively. The lower limit of quantitation for this assay was 0.01 µM. The differences in LC/MS performance were investigated by conducting a comparison of UPLC with another method previously optimized for HPLC‐based separation and quantification of testosterone and its metabolites. Copyright © 2006 John Wiley & Sons, Ltd.