气相色谱-质谱联用测定大鼠脑部的神经甾体

Determination of Neurosteroids in Rat Brain by Gas Chromatography/Mass Spectrometry

应用气相色谱 质谱联用技术建立了大鼠脑部神经甾体的测定方法。游离型甾体和甾体硫酸酯分两步萃取。第一步用乙酸乙酯提取游离型甾体,第二步用氯仿/2 丁醇提取甾体硫酸酯,然后经固相萃取纯化。甾体硫酸酯进行溶剂解形成游离型甾体。游离型甾体和甾体硫酸酯分别经七氟丁酸酐衍生化后进行气相色谱 质谱分析。经初步研究雄性大鼠脑部游离型神经甾体孕烯醇酮(PREG)、黄体酮(PROG)、别孕烯醇酮(AP)和脱氢表雄酮(DHEA)的含量分别为(8 53±1 11)ng/g,(7 01±2 60)ng/g,(1 17±0 19)ng/g,(0 92±0 20)ng/g。甾体硫酸酯孕烯醇酮硫酸酯(PREGS)和脱氢表雄酮硫酸酯(DHEAS)的含量分别为(5 94±2 03)ng/g和(1 93±0 92)ng/g。每一种神经甾体都有好的线性关系和准确度。该法适合同时测定大鼠脑部游离型和结合型甾体。

A simplified method has been established to simultaneously characterize different neurosteroids in rat brain by gas chromatography/mass spectrometry (GC-MS). Neurosteroids were isolated separately in a two-step procedure using ethyl acetate in the first step to extract the unconjugated steroids and chloroform/2-butanol(50/50,v/v) in the second step to extract sulfated steroids. All steroid fractions were further purified by solid phase extraction (SPE) and the sulfated steroids were solvolyzed. All steroids were derivatized with heptafluorobutyric acid anhydride and analyzed by GC-MS (electrospray ionization) using selected ion monitoring. In male rat brain, the concentrations of pregnenolone (PREG), progesterone (PROG), allopregnanolone (AP) and dihydroepiandrosterone (DHEA) were (8.53±1.11) ng/g, (7.01±2.60 )ng/g, (1.17±0.19 )ng/g, and (0.92±0.20)ng/g, respectively. The concentrations of pregnenolone sulfate (PREGS) and dihydroepiandrosterone sulfate (DHEAS) were (5.94±2.03) ng/g and (1.93±0.92) ng/g, respectively. Good linearity and accuracy were observed for each steroid. The procedure was suitable for measuring the concentrations of endogenous neurosteroids, simultaneously including the sulfates in rat brain .