Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry.
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.
Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N Wolfe St, Baltimore, MD 21205-2179, USA.
Alissa
A
Renne
Lindi
L
Luo
Jonathan
J
Jarow
William W
WW
Wright
Terry R
TR
Brown
Haolin
H
Chen
Barry R
BR
Zirkin
Marlin D
MD
Friesen
D000818:Animals; D001826:Body Fluids / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium; D007201:Indicator Dilution Techniques; D008297:Male; D051381:Rats; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods; D013737:Testis / Q000737:chemistry*; D013739:Testosterone / Q000032:analysis*
D013739:Testosterone; D003903:Deuterium