title: Methods for differential and quantitative analyses of brain neurosteroid levels by LC/MS/MS with ESI-enhancing and isotope-coded derivatization.
abstract: The analysis of changes in the brain neurosteroid (NS) levels due to various stimuli can contribute to the elucidation of their physiological roles, and the discovery and development of new antipsychotic agents targeting neurosteroidogenesis. We developed methods for the differential and quantitative analyses of the brain levels of allopregnanolene (AP) and its precursor, pregnenolone (PREG), using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using 2-hydrazino-1-methylpyridine (HMP) and its isotope-coded analogue, (2)H3-HMP (d-HMP). For the differential analysis, the brain sample of an untreated rat was derivatized with HMP, while the brain sample of a treated (stressed or drug-administered) rat was derivatized with d-HMP. The two derivatives were mixed and then subjected to LC/ESI-MS/MS. The stress- and drug (clozapine and fluoxetine)-evoked increases in the brain AP and PREG levels were accurately analyzed by the developed method. It was also possible to determine the absolute concentrations of the brain steroids when a deuterium-coded moiety was introduced to the standard steroids of known amounts by the derivatization and the resulting derivatives were used as internal standards. The HMP-derivatization enabled the highly sensitive detection and the use of d-HMP significantly improved the assay precision [the intra- (n=5) and inter-assay (n=5) relative standard deviations did not exceed 13.7%] and accuracy (analytical recovery ranged from 98.7 to 106.7%).
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address: higashi@rs.tus.ac.jp.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Higashi
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Aiba
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Yoshizawa
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Shoujiro
authors.initials: S
authors.lastname: Ogawa
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D007553:Isotope Labeling / Q000379:methods / Q000592:standards*; D008297:Male; D018377:Neurotransmitter Agents / Q000032:analysis* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods / Q000592:standards*; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*
chemical_list: D018377:Neurotransmitter Agents
keywords: Antipsychotic drug; Isotope-coded derivatization; LC/ESI–MS/MS; Neurosteroid; Rat brain