高效液相色谱-荧光法测定大鼠纹状体细胞外液中4种氨基酸的含量

目的: 采用高效液相色谱-荧光检测法(HPLC-RF)建立大鼠脑微透析液中天门冬氨酸(Asp)、谷氨酸(Glu)、甘氨酸(Gly) 和γ-氨基丁酸(GABA)的测定方法。 方法: 采用ODS-C₁₈(5 μm,250 mm × 4.6 mm)色谱柱,柱温为35℃,流动相A为磷酸盐缓冲液(pH 6.7),流动相B为纯甲醇,梯度洗脱,流速为1.0 mL·min^-1,荧光检测器的激发波长为239 nm,发射波长为375 nm。以邻苯二甲醛和β-巯基乙醇为衍生化试剂,柱前衍生,将脑微透析液直接注入高效液相色谱仪完成分析。 结果: Asp、Glu、Gly和GABA 4种氨基酸在0.5~32 μmol·L^-1范围内线性关系良好,回收率范围为96.2%~102.1%。 结论: 所建立的氨基酸检测方法具有流动相配制简单、保留时间稳定、分析时间短、重复性好和回收率高等优点,可用于脑微透析液中痕量氨基酸的测定。

Objective: To develop an HPLC-RF method for Asp,Glu,Gly and GABA determination in rat cerebral microdialysates. Methods: ODS-C₁₈(5 μm,250 mm × 4.6 mm) column was employed by a gradient elution,using phosphate buffer solution (pH 6.7) as mobile phase A and pure methanol as mobile phase B at the column temperature of 35℃ and the flow rate of 1 mL·min^-1.Linear gradient elution was adopted with a florescent detector(λ_ex=239 nm,λ_em=375 nm).O-phthaldialdehyde/β-mercaptoethanol was used for the derivatization of amino acids.Then HPLC-RF was used to measure the striatal extracellular levels of four amino acids. Results: These four components were all found to have good linear relationship over a concentration range of 0.5-32 μmol·L^-1 ,and the recoveries of four amino acids were in the range of 96.2%-102.1%. Conclusions: The HPLC-RF method has the advantages of simple preparation,constant retention time,short analysis time,good repeatability and high recovery rate,which can be used for cerebral microdialysate trace determination of amino acids.