高效液相色谱-质谱法测定大鼠不同脑区的硫酸酯型神经甾体

Determination of Neurosteroids in Rat Brain Regions by Liquid Chromatography/Negative Atomspheric Pressure Ionization Mass Spectrometry

应用高效液相色谱 质谱联用技术,以雌酮硫酸酯为内标,建立了大鼠不同脑区硫酸酯型神经甾体的测定方法。甾体分两步萃取,第一步用氯仿 仲丁醇(体积比为1∶1)提取甾体硫酸酯,然后经固相萃取纯化。溶剂解使甾体硫酸酯形成游离型甾体,然后用衍生化试剂进行衍生化,再用液相色谱 质谱分离测定。初步研究发现,雄性SD大鼠不同脑区神经甾体孕烯醇酮硫酸酯和脱氢表雄酮硫酸酯的含量分别为(4 14±1 33)ng/g和(2 26±0 76)ng/g(垂体),(1 98±1 13)ng/g和(1 80±0 93)ng/g(下丘脑),(1 08±0 48)ng/g和(0 81±0 23)ng/g(额叶皮质),(0 72±0 19)ng/g和(0 77±0 12)ng/g(海马),(1 70±0 45)ng/g和(1 44±0 71)ng/g(杏仁核),(0 92±0 27)ng/g和(0 85±0 44)ng/g(纹状体),(3 62±1 77)ng/g和(3 17±2 11)ng/g(伏隔核)。每种神经甾体与内标的峰面积比和甾体浓度都有良好的线性关系,测定准确度较高。该方法可满足大鼠不同脑核团内结合型甾体的分离测定。

A simplified method has been established using liquid chromatography-negative atomspheric pressure ionization mass spectrometry (LC-MS) to simultaneously determine two conjugated neurosteroids from rat brain regions. Neurosteroids were separately isolated in a two-step procedure using chloroform/2-butanol (50∶50, v/v) where the first step was to extract sulfated steroids, then steroid fractions were purified by solid phase extraction (SPE), and finally the sulfated steroid was solvolyzed. Estrogen sulfate was chosen as internal standard. All steroids were derivatized with 2-nitro-4-trifluoromethylphenylhydrazine (2NFPH) and analyzed by LC-MS using selected-ion monitoring. LC-MS was performed on an Agilent 1100 liquid chromatograph-mass spectrometer with atmospheric pressure chemical ionization (APCI) interface, and the hydrazones of oxosteroids was analyzed in a negative-ion mode. A Zorbax SB C_(18) column was used with a flow rate of 1 mL/min at 40 ℃. The mobile phase consisted of acetonitrile and distilled water. The concentrations of PREGS and DHEAS in male rat brain regions were (4.14±1.33) (ng/g) and (2.26±0.76) (ng/g) (pituitary gland), (1.98±1.13) (ng/g) and (1.80±0.93) (ng/g) (hypothalamus), (1.08±0.48) (ng/g) and (0.81±0.23) (ng/g) (frontal cortex), (0.72±0.19) (ng/g) and (0.77±0.12) (ng/g) (hippocampus), (1.70±0.45) (ng/g) and (1.44±0.71) (ng/g) (amygdale), (0.92±0.27) (ng/g) and (0.85±0.44) (ng/g) (striatum), (3.62±1.77) (ng/g) and (3.17±2.11) (ng/g) (nucleus accumbens), respectively. Good linearity and accuracy were observed for each steroid. The procedure was suitable for measuring concentrations of the sulfated steroids in rat brain regions simultaneously.