柱前衍生-HPLC测定大鼠海马组织中的谷氨酸和瓜氨酸

Determination of glutamate and citrulline in rat hippocampus by a pre-column derivative HPLC method

目的 采用HPLC法测定大鼠脑内海马组织中谷氨酸和瓜氨酸的浓度。方法 取20只大鼠的海马组织,经低温匀浆处理,利用邻苯二甲醛柱前衍生,采用Diamonsil RP-C_（18）柱（250 mm×4.6 mm,5μm）,流动相为甲醇-乙腈-0.05mol·L^（-1）乙酸钠（乙酸调pH6.0）水溶液进行梯度洗脱,流速0.8 mL·min^（-1）,柱温30℃,荧光检测λ_（ex）=334 nm、λ_（em）=456 nm,内标为β-丙氨酸。结果 0.5~100μmol·L^（-1）谷氨酸和瓜氨酸与峰面积的线性关系良好（r^2分别为0.998、0.991）,最低检测限均为0.1μmol·L^（-1）,低、中、高浓度谷氨酸和瓜氨酸的加样回收率分别为90.6%、96.9%、98.8%和95.3%、97.9%、91.5%。日内和日间精密度的RSD〈8.0%。结论 所用方法准确简便,可用于大鼠海马组织中谷氨酸和瓜氨酸的浓度分析。

OBJECTIVE To determine glutamate and citrulline in rat hippocampus by HPLC method. METHODS The hippocam- pus of 20 rats were removed and dissected out on an ice - cold plat. The glutamate and citrulline were derivatived with o - phthalalde- hyde and analyzed by HPLC - fluorescence. Diamonsil RP - Cls column （ 250 mm Ф 4.6 mm, 5 μm） was used. The mobile phase consisted of methanol - acetonitrile -0.05 mol L-1 potassium acetate water （pH was adjusted to 6.0 with acetic acid） at the flow rate of 0.8 mL. min-1 by gradient elution. The column temperature was maintained at 30 ℃. The Aox was 334 nm ,Aem was 456 nm. The β - Alanine was used as the internal standard. RESULTS A good linear relationship was obtained between the concentration of glutamate and eitrnlline from 0.5 μmol L-1 to 100 μmol L-1 （r2 =0. 998 and 0. 991 ）. The limit of detection for the analysis of glutamate and citrnlline both were 0.1 μmol L-1. The sample recovery rates of glutamate were 90.6% ,96.9% , and 98.8% and of eitrnlline were 95.3% ,97.9% , and 91.5% for low, middle and high three concentrations, respectively. The intra - day and inter - day precision （RSD） were all less than 8.0%. CONCLUSION The present method is accurate and simple which could be used to determine gluta- mate and citrnlline in rat hippocampus. It can be used in the clinical pharmacology study of glutamate and citrnlline.