Liquid chromatography–tandem mass spectrometry assay for human serum testosterone and trideuterated testosterone

A liquid chromatography tandem mass spectrometry assay for serum testosterone (T) and trideuterated testosterone (d3T) was developed in order to support clinical research studies that determine the pharmacokinetics, production rate, and clearance of testosterone by administration of trideuterated testosterone. After adding 19-nortestosterone as the internal standard (I.S.), sodium acetate buffer, and ether, to a serum aliquot, the mixture was shaken and centrifuged, and the ether was dried. The extract was reconstituted in methanol and 15 μl was injected into a liquid chromatograph equipped with an autosampler and Applied Biosystems-Sciex API 300 triple quadrupole mass spectrometer operated in the positive ion mode. T, d3T, and I.S. were monitored with transitions m/z 289 to m/z 97, m/z 292 to m/z 97, and m/z 275 to m/z 109, respectively. The two calibration curves were linear over the entire measurement range of 0–20 ng/ml for T and 0–2.0 ng/ml for d3T. The LOQs for T and d3T were 0.5 ng/ml and 0.05 ng/ml. The recoveries for T and d3T were 91.5 and 96.4%. For T at 1.25 ng/ml and 4.0 ng/ml, the intra-day precision (RSD, %) was 3.9 and 4.3% and intra-day accuracy 0.01 and 4.5%, respectively. The inter-day precision at these levels was 5.3 and 5.4% and inter-day accuracy was 1.9 and 0.3%. For d3T at 0.125 ng/ml and 0.4 ng/ml, the intra-day precision (RSD, %) was 2.8 and 8.3% and intra-day accuracy was 1.8 and 5.6%. The inter-day precision at these levels was 10.0 and 7.6% and inter-day accuracy was 5.7 and 3.4%. The concentrations of T in the 38 healthy subjects ranged from 2.5 to 14.0 ng/ml (mean 6.2 ng/ml).