Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry

Abstract

An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17β-estradiol (E(2)), and 5α-androstan-3α,17β-diol (3α-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-μL samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E(2). Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E(2) and 3α-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.

Figure 1

Analytical scheme for analysis of testosterone, dihydrotestosterone, 17β-estradiol, and 5α-androstan-3α,17β-diol in rat testicular fluid. HPLC-MS/MS indicates high-performance liquid chromatography–tandem mass spectrometry; SPE, solid phase extraction.

Figure 2

High-performance liquid chromatography–tandem mass spectrometry mass chromatograms for the analysis of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17β-estradiol (E₂), and 5α-androstan-3α,17β-diol (3α-diol) in testicular fluid. Structures are shown only for deuterium-labeled compounds. D₃ indicates tri-deuterium–labeled (16,16,17-D₃).

Figure 3

Correlation (r² = .98) for the analysis of testosterone by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) and radioimmunoassay (RIA). The slope of the correlation curve is y = 1.07 + 9.37.

Figure 4

Correlation of testosterone and dihydrotestosterone in the intratesticular fluid of individual rats. HPLC-MS/MS indicates high-performance liquid chromatography–tandem mass spectrometry.