全脑缺血-再灌注大鼠脑组织中DADLE浓度的测定

Determination of[D-Ala^2,D-Leu^5]Enkephalin Concentration in Brain Tissues of Rats with Global Cerebral Ischemia Reperfusion

目的建立快速、准确检测大鼠脑组织中[d-丙氨酸2,d-亮氨酸5]脑啡肽(DADLE)的液相色谱-串联质谱(LC-MS/MS)定量检测方法。方法SD大鼠脑组织样品匀浆,乙腈沉淀蛋白,C18反相色谱柱(2.0 mm×50 mm,5μm)以水-甲醇(0.1%甲酸)体系为流动相,室温下,流速0.4 mL·min^-1,经LC-MS/MS系统进行定量分析。全脑缺血-再灌注模型大鼠分别单次颈静脉注射DADLE 2,5,10 mg·kg^-1后,于10,20 min测定脑组织中DADLE浓度。结果SD大鼠脑组织匀浆液质量浓度在0.1~1000 ng·mL^-1线性关系良好,定量下限为0.1 ng·mL^-1,批内、批间精密度RSD<11.6%,准确度范围95.81%~99.19%。缺血-再灌注大鼠给药后10 min脑组织DADLE浓度差异不显著,不同剂量给药后20 min脑组织浓度分别为(1.3±0.45),(2.2±1.1),(2.9±1.4)ng·mL^-1。结论该分析方法快速、简单,成功应用于DADLE脑组织样品检测。

Objective To develop a LC-MS/MS method for determination DADLE in brain tissue of rats.Methods The rats’brain homogenates were precipitated by acetonitrile.And the separation was carried out by a reversal C18(2.0 mm×50 mm,5μm)column in water-menthol system with 0.1%formic acid,the flow rate was 0.4 mL·min-1 followed by mass spectrometry quantitation.Rats with global cerebral ischemia reperfusion were injected with 2,5,10 mg·kg^-1 DADLE through jugular vein.And the concentrations in brain tissue were tested 10 and 20 minutes after injection.Results DADLE in rat brain tissue has a good linearity from 0.1 to 1000 ng·mL^-1 with the LOQ of 0.1 ng·mL^-1.Both the intra-and inter-batch precisions of RSD were within 11.6%.And the accuraues were in the range of 95.81%-99.19%.DADLE concentrations in rats'brain tissue were not varied significantly 10 minutes after injection,and were(1.3±0.45),(2.2±1.1),(2.9±1.4)ng·mL^-120 minutes after injection of 2,5,10 mg·kg^-1 DADLE.Conclusion A rapid and simple quantitation method of DADLE is well established and validated,which applied to determination of the DADLE concentration in rat brain tissue.