Determination of salivary testosterone and androstendione by liquid chromatography–tandem mass spectrometry

BackgroundSalivary steroid measurements have the potential to provide a non-invasive assessment of serum free steroid concentrations. Salivary testing has been proposed as a possible substitute for serum testing partly because of easy sample collection.MethodsWe developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for simultaneous measurement of testosterone and androstendione in saliva. We spiked 100 μL of saliva with isotope-labeled internal standards and performed extraction with diethyl ether. After evaporation both steroids were analyzed by LC–MS/MS operating in the positive mode electrospray ionization (ESI) after separation on a reversed-phase column.ResultsThe calibration curves for analysis of testosterone and androstendione exhibited consistent linearity and reproducibility in the range of 20–1000 pmol/L. The limit of quantitation (LOQ) was 10 pmol/L for both steroids. The mean recovery of the analytes added to saliva ranged from 97 to 102%. Between-run CVs were 3.6–4.0% for testosterone and 4.8–8.0% for androstendione. The upper limit (the 97.5 percentile) of testosterone and androstendione reference ranges for males was 356 pmol/L and 344 pmol/L and that for females 39 pmol/L and 349 pmol/L, respectively.ConclusionsThis LC–MS/MS method has the required lower limit of quantification for analysis of salivary concentrations of testosterone and androstendione. The assay is rapid, reliable and simple to perform with a routine LC–MS/MS spectrometer with required sensitivity.

► Saliva is a possible substitute for serum because of easy sample collection.
► We developed an LC–MS/MS assay for testosterone and androstendione in saliva.
► The limit of quantitation (LOQ) was 10 pmol/L for both steroids.
► Reference ranges for males and females were determined.
► The assay is simple to perform with a routine LC–MS/MS with required sensitivity.