Saliva contains the free unbound fraction of testosterone, which has been shown to correlate with the free circulating serum testosterone, and may better reflect the physiologically active form. Recently, measurement of free testosterone has gained international recommendations for the diagnosis of hypogonadism in the ageing male. Saliva collection is a non-invasive technique, offering more flexibility to the patient, e.g. the collection of samples at home. Liquid chromatographytandem mass spectrometry (LCMS/MS) offers a specific, sensitive technique for measuring this analyte.
Saliva (200 μl) and D5-testosterone internal standard (10 μl) were extracted with 1 ml methyl-tert-butyl-ether. The supernatant was evaporated and reconstituted with 100 μl of 50:50 mobile phase; water (A) and methanol (B), each containing 2 mmol/l ammonium acetate, and 0.1% (v/v) formic acid. Extract (30 μl) was injected onto an ACQUITY HSS T3 2.1×50 mm, 1.8 μm column. Testosterone and D5-testosterone were eluted from the column at 1.3 min using an increasing linear gradient of 5090% mobile phase B over 1.5 min. The column was then washed with 98% B for 0.75 min followed by re-equilibration with starting conditions. The flow rate was maintained at 0.6 ml/min throughout and the total run time was 3.5 min injection to injection. The transitions for testosterone and D5-testosterone were m/z 289.2>108.8 and m/z 294.1>99.8.
The lower limit of quantitation was 25 pmol/l, was linear to at least 52 nmol/l. The mean recoveries were 96, 100 and 96% at 0.15, 0.57 and 2.23 nmol/l respectively. Intra-assay and inter-assay imprecision were 8.4, 6.2, 3.5 and 4.3% and 7.8, 5.9, 2.2 and 2.4% respectively at concentrations of 0.1, 0.25, 1.0 and 4.0 nmol/l.
We have developed a simple, robust assay, which has sufficient sensitivity to measure testosterone in male saliva. This assay may be used to assess condition such as hypogonadism in males.