Quantitative analysis of eight testosterone metabolites using column switching and liquid chromatography/tandem mass spectrometry

The rate at which testosterone is metabolized to different singly hydroxylated metabolites has been widely used as an in vitro marker for activity of different CYP450 enzymes. The interest in extra‐hepatic metabolism, e.g. due to metabolism in the gut wall, has increased during the last decade. Measurement of extra‐hepatic enzyme activity using testosterone as a substrate requires a highly sensitive analytical method. A new liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using column switching for online cleaning and desalting of samples, was developed and validated for analysis of 2α‐, 2β‐, 6α‐, 6β‐, 7α‐, 16α‐, and 16β‐hydroxytestosterone and androstenedione. The samples were injected on a SB‐CN column and detection was performed using MS/MS. The limits of quantification ranged from 0.3 to 3.33 nM for the different metabolites. The validated method was used to quantify the enzyme activity in rat intestine mucosa. The formation rates of 16α‐, 16β‐hydroxytestosterone and androstenedione were quantified, and 2β‐and 6β‐hydroxytestosterone were formed above the limits of detection. Copyright © 2004 John Wiley & Sons, Ltd.