Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry

A rapid, sensitive, and specific method using liquid chromatography tandem mass spectrometry (LC/MS/MS) has been developed for simultaneous determination of testosterone (T), dihydrotestosterone (DHT), estradiol (E2), and 5α-androstan-3α, -17β-diol (3α-Diol) within human testicular fluid. Sample pretreatment involved a one-step extraction with diethyl ether. The analytes were separated on a Waters X-Terra™ C ₁₈ (150 mm × 2.1 mm i.d., 3.5 μm) analytical column with acetonitrile/water mobile phase (70:30, v/v) containing 0.1% formic acid using isocratic flow at 0.15 ml/min for 8 min. The column effluent was monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.1-50 ng/ml for T, 0.02-1 ng/ml for DHT, 0.05-2 ng/ml for E2, and 0.2-10 ng/ml for 3α-Diol, with values for the coefficient of determination of >0.99. The overall extraction efficiency was greater than 86% for T, 75% for DHT, 66% for E2, and 60% for 3α-Diol. The values for within-day and between-day precision and accuracy were <15%. We measured each of the four steroids in testicular sample volumes of only 20 μl, obtained by percutaneous testicular aspiration. The mean intratesticular testosterone concentration found by LC/MS/MS, 572 ± 102 ng/ml, was similar to that previously obtained by radioimmunoassay (RIA). The mean intratesticular estradiol concentration was 15.7 ± 2.3 ng/ml, which also correlated well with RIA measurement. Both DHT and 3α-Diol were below the limits of detection by RIA, but could be measured accurately by LC/MS/MS. In conclusion, LC/MS/MS represents a sensitive and accurate means by which to measure four separate steroids within small volume samples of testicular fluid.