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 5alpha-androstan-3alpha, -17beta-diol (3alpha-Diol) within human testicular fluid. Sample pretreatment involved a one-step extraction with diethyl ether. The analytes were separated on a Waters X-Terra C18 (150 mm x 2.1 mm i.d., 3.5 microm) 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 3alpha-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 3alpha-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 microl, 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 3alpha-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.
Zhao
Ming
M
Division of Experimental Therapeutics, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Baker
Sharyn D
SD
Yan
Xiaohua
X
Zhao
Yiyu
Y
Wright
William W
WW
Zirkin
Barry R
BR
Jarow
Jonathan P
JP
D015113:Androstane-3,17-diol / Q000032:analysis; D002138:Calibration; D002845:Chromatography; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D013196:Dihydrotestosterone / Q000032:analysis; D004958:Estradiol / Q000032:analysis; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D011863:Radioimmunoassay; D021241:Spectrometry, Mass, Electrospray Ionization; D013256:Steroids / Q000032:analysis*; D013737:Testis / Q000378:metabolism*; D013739:Testosterone / Q000032:analysis / Q000378:metabolism; D013997:Time Factors; D014867:Water
D013256:Steroids; D014867:Water; D013196:Dihydrotestosterone; D015113:Androstane-3,17-diol; D013739:Testosterone; D004958:Estradiol