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.

Figure 1

Proposed scheme for the formation of T-HTP via reaction of HTP with T.

Figure 2

Product ion spectra of T-HTP (A) and DHT-HTP (B) derivatives. Proposed fragmentation patterns of the [M+H]⁺ ion of the T-HTP (A) and DHT-HTP (B) derivatives under CID are also shown.

Figure 3

MS/MS ion chromatograms for HTP derivatives of T (A, 449/257), DHT (B, 451/260), and their internal standards MT (C, 463/257) and MDHT (D, 465/274), obtained from medium QC samples. The minor peaks in panels B and D represent background signals (not produced by the standard).

Figure 4

MS/MS ion chromatograms for T-HTP and DHT-HTP in blank CS-FBS (A and C) and LLOQ samples (B and D).