Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry.
A novel method was established for simultaneous quantitation of testosterone (T) and dihydrotestosterone (DHT) in murine tissue and serum samples. Endogenous T and DHT, together with the internal standards 17alpha-methyl-T and 17alpha-methyl-DHT, were extracted from tissues and then derivatized by reaction with 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP). Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) resulted in product ion spectra of HTP derivatives of both T and DHT that showed analyte-specific fragmentations; the latter fragmentations were characterized by the use of high-resolution Orbitrap MS/MS. These specific fragmentations enabled quantitation of T and DHT in the multiple-reaction monitoring (MRM) mode. The method was validated with charcoal-stripped serum as the matrix. The lower limit of quantitation (LLOQ) was 0.10ng/ml for T and 0.50ng/ml for DHT. The method was then used for determination of serum and tissue levels of T and DHT in transgenic mice carrying a hypomorphic NADPH-cytochrome P450 reductase gene (Cpr-low mice). Remarkably, ovarian T levels in Cpr-low mice were found to be 25-fold higher than those in wild-type mice, a finding that at least partly explains the female infertility seen in the Cpr-low mice. In conclusion, our method provides excellent sensitivity and selectivity for determination of endogenous levels of T and DHT in mouse tissues.
Weng
Yan
Y
Wadsworth Center, New York State Department of Health, State University of New York at Albany, 12201, USA.
Xie
Fang
F
Xu
Li
L
Zagorevski
Dmitri
D
Spink
David C
DC
Ding
Xinxin
X
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D013196:Dihydrotestosterone / Q000097:blood* / Q000378:metabolism*; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013739:Testosterone / Q000097:blood* / Q000378:metabolism*; D014018:Tissue Distribution
D013196:Dihydrotestosterone; D013739:Testosterone; D009251:NADPH-Ferrihemoprotein Reductase