论文部分内容阅读
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。
Objective To establish a method for the determination of glutamic acid in rat cerebrospinal fluid by high performance liquid chromatography. Methods Cerebrospinal fluid samples collected from microdialysis experiments were derivatized with o-phthalaldehyde and β-mercaptoethanol and separated by high performance liquid chromatography. The mobile phase A was 50 mmol.L-1 sodium acetate (adjusted to pH 6.7 with acetic acid) and the mobile phase B was methanol / tetrahydrofuran (97.5 / 2.5, v / v) The program was (T, B%) (0,30) (7,45) (14,30) (20,30), the flow rate was 1.0 mL.min-1, quantified by fluorescence detector with excitation wavelength of 340 nm and emission wavelength of 450 nm. Perfusion of high potassium cerebrospinal fluid to investigate the changes of glutamate in the striatum of rats. Results The linear range of glutamic acid was 3.12 × 10-2 ~ 1.00 mg.L-1 (γ = 0.9998). The intra-day and inter-day precision were all less than 15%, and the recoveries of low, middle and high concentrations were 96.6 ± 6.1)%, (99.1 ± 7.7)%, (96.2 ± 4.1)%. After high potassium artificial cerebrospinal fluid perfusion, glutamate significantly increased and then decreased, followed by an upward trend. Conclusion The method is accurate, stable and sensitive and suitable for the quantitative analysis of glutamate in microdialysis samples.