Studies on neurosteroids XX. Liquid chromatography-tandem mass spectrometric method for simultaneous determination of testosterone and 5alpha-dihydrotestosterone in rat brain and serum.
Testosterone (T) and 5alpha-dihydrotestosterone (DHT) are now referred to not only as androgenic steroid hormones, but also as neuroactive steroids, because they elicit anesthetic and anxiolytic effects. Methods using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) coupled with derivatization are developed and validated to examine rat brain and serum levels of T and DHT and their stress-induced changes. The steroids are extracted with methanol-acetic acid from the brain tissue or serum, purified using solid-phase extraction cartridges, derivatized with a permanently charged reagent, 2-hydrazino-1-methylpyridine, and subjected to LC-MS-MS. [19,19,19-(2)H3]-T is used as the internal standard. The intra- and inter-assay coefficients of variation are below 10.0%, and the analytical recoveries are 98.1-103.0%. The developed methods are applied to the animal study and it was found that a fair amount of DHT is continuously and locally synthesized in the brain, and its level is not changed by the immobilization stress and depends on the brain T level.
Higashi
Tatsuya
T
Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Ninomiya
Yasuaki
Y
Shimada
Kazutake
K
D015068:17-Ketosteroids / Q000032:analysis* / Q000097:blood; D000732:Androstanols / Q000032:analysis* / Q000097:blood; D000818:Animals; D001923:Brain Chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012149:Restraint, Physical; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013739:Testosterone / Q000032:analysis* / Q000097:blood
D015068:17-Ketosteroids; C418023:5-dihydrorubrosterone; D000732:Androstanols; D013739:Testosterone