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

Ming Zhao, Sharyn D. Baker, Xiaohua Yan, Yiyu Zhao, William W. Wright, Barry R. Zirkin, Jonathan P. Jarow

Research output: Contribution to journalArticlepeer-review

47 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)721-726
Number of pages6
Issue number11-12
StatePublished - Oct 2004


  • Dihydrotestosterone
  • Estradiol
  • Mass spectrometry
  • Testes
  • Testosterone

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology
  • Pharmacology
  • Clinical Biochemistry
  • Organic Chemistry


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