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

Salivary 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.We 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.The 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.This 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.