Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Abstract

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.

• Authors: Yan Weng, Fang Xie, Li Xu, Dmitri Zagorevski, David C Spink, Xinxin Ding
• Journal: Analytical biochemistry (Anal Biochem) Vol. 402 Issue 2 Pg. 121-8 (Jul 15 2010) ISSN: 1096-0309 [Electronic] United States
• PMID: 20361922 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Validation Study)
• Copyright: 2010 Elsevier Inc. All rights reserved.

Chemical References

• Dihydrotestosterone
• Testosterone
• NADPH-Ferrihemoprotein Reductase

Topics

• Animals
• Chromatography, Liquid (methods)
• Dihydrotestosterone (blood, metabolism)
• Female
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Transgenic
• NADPH-Ferrihemoprotein Reductase (genetics)
• Spectrometry, Mass, Electrospray Ionization (methods)
• Testosterone (blood, metabolism)
• Tissue Distribution