title: Studies on neurosteroids XXIII. Analysis of tetrahydrocorticosterone isomers in the brain of rats exposed to immobilization using LC-MS.
abstract: The identification and quantification of tetrahydrocorticosterone isomers (THBs; 3alpha,5alpha-, 3beta,5alpha-, 3alpha,5beta- and 3beta,5beta-THB) in rat brains using liquid chromatography (LC)-mass spectrometry (MS) are described. For the identification, the THBs were converted to the atmospheric pressure chemical ionization (APCI)-active derivatives, i.e., the dinitrobezoyl esters and 2-nitro-4-trifluoromethylphenyl hydrazones, and detected in the negative-ion mode. These derivatives showed 60- and 40-fold higher sensitivities, respectively, than intact steroids measured in the positive-APCI-MS. The derivatized THBs were satisfactorily separated from the others during the reversed-phase LC. The THBs were not detected at all in the brains of the unstressed rats. When the rats were exposed to the immobilization for 20 min, 3alpha,5alpha- and 3beta,5alpha-THB were detected as the major metabolites together with small amounts of 3alpha,5beta- and 3beta,5beta-THB in the male rat brain, while only 3alpha,5alpha-THB was detected in the female rats. Thus, the steroid variety found in the brains was different between the sexes. In the next step, 3alpha,5alpha-THB, a major metabolite found in the brains of the stressed rats, was quantified as its dinitrobezoyl ester. This method was accurate and reproducible, and the limit of quantitation was 1.0 ng/g tissue when a 50 mg tissue sample was used. There was also a sex difference in the brain 3alpha,5alpha-THB level; it was significantly higher in the female rats than in the male rats (P<0.05), although the brain corticosterone level was not higher in the stressed female rats than in the male rats (no statistical difference).
authors.affiliation: Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. higashi@p.kanazawa-u.ac.jp
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Higashi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yokoi
authors.forename: Hitoe
authors.initials: H
authors.lastname: Maekubo
authors.forename: Ayako
authors.initials: A
authors.lastname: Honda
authors.forename: Kazutake
authors.initials: K
authors.lastname: Shimada
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D003345:Corticosterone / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D007103:Immobilization; D007536:Isomerism; D013058:Mass Spectrometry; D051381:Rats; D017208:Rats, Wistar; D013256:Steroids / Q000032:analysis
chemical_list: D013256:Steroids; C003676:tetrahydrocorticosterone; D003345:Corticosterone