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

作者：Weng Yan; Xie Fang; Xu Li; Zagorevski Dmitri; Spink David C; Ding Xinxin^*

来源：Analytical Biochemistry, 2010, 402(2): 121-128.

DOI：10.1016/j.ab.2010.03.034

• LC-MS
• Steroids
• Testosterone
• Dihydrotestosterone
• Metabolism
• Cytochrome P450
• Mice

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 17 alpha-methyl-T and 17 alpha-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 0.10 ng/ml for T and 0.50 ng/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.

• 出版日期2010-7-15