高效液相色谱法测定人工脑脊液中谷氨酸的浓度

目的建立高效液相色谱测定大鼠脑脊液中谷氨酸含量的方法。方法微透析实验收集到的脑脊液样品用邻苯二甲醛、β巯基乙醇衍生化反应后,高效液相色谱法分离。用RP18色谱柱,流动相A为50 mmol.L-1乙酸钠(乙酸调pH为6.7),流动相B为甲醇/四氢呋喃(97.5/2.5,v/v),用二元梯度洗脱,梯度程序为(T,B%)(0,30)(7,45)(14,30)(20,30),流速1.0 mL.min-1,荧光检测器定量,激发波长340 nm,发射波长450 nm。用高钾脑脊液灌流,考察大鼠纹状体内谷氨酸的变化。结果谷氨酸的线性范围为3.12×10-2～1.00 mg.L-1(γ=0.9998),日内、日间精密度均<15%,低、中、高浓度回收率分别为(96.6±6.1)%,(99.1±7.7)%,(96.2±4.1)%。高钾人工脑脊液灌流后,谷氨酸明显升高后降低,随后又有升高的趋势。结论本方法准确,稳定,灵敏,适用于微透析样品中谷氨酸的定量分析。

Development of HPLC method to measure glutamic acid in artificial cerebrospinal fluid

Objective To develop a sensitive,accurate and specific high performance liquid chromatography(HPLC) method to measure concentration of glutamic acid in rat cerebrospinal fluid.MethodsMicrodialysis samples were derivatized by o-phthaldialdehyde(OPA) and β-mercaptoethanol,separated by HPLC and quatitated by fluorescence detector.Mobile phase A was 0.05 mol·L-1 sodium acetate(pH adjusted to 6.7 with acetic acid),mobile phase B was methanol/tetrahydrofuran(97.5/2.5,v/v).Gradient program was(T,B%)(0,30)(7,45)(14,30)(20,30).The total flow rate was 1.0 mL·min-1.Excitation wave was 340 nm,and emission wave was 450 nm.Investigating changes of glutamic acid in rat′s striatum was used high potassium artificial cerebrospinal fluid(aCSF) perfusion through microdialysis probe.ResultsThe retention time of glutamic acid was 2.8 min.Good linearity(γ=0.9998) was obtained between 3.12×10-2 and 1.00 mg·L-1.RSD of intra-day and inter-day validation was lower than 15%.Recovery results of QC were(96.6±6.1)%,(99.1±7.7)%,(96.2±4.1)%,respectively.After perfusion of high potassium aCSF,glutamic acid increased significantly and then decreased to baseline,and there was a trend of increasing after that.ConclusionThe method is sensitive,accurate and specific,and can be applied to the quantification of glutamic acid in microdialysis samples of rat.