液相色谱法测定小鼠脑组织中ATP、ADP及AMP含量

目的建立小鼠脑组织中腺苷酸含量测定的高效液相色谱(high performance liquid chromatogram,HPLC)-可变波长检测法。方法应用酸沉淀蛋白、Agilent1200高效液相色谱系统、Supelco Discovery C18(150 mm×4.6 mm,5μm)反相色谱柱及相同填充材料的预柱,对小鼠脑组织样品中腺苷酸进行分离分析,波长为254 nm,流动相为30 mmol/L K2HPO4-KH2PO4(含5%甲醇),pH5.85,流速为1.0 mL/min,进样体积为20μL,柱温为30℃。结果腺苷酸ATP、ADP及AMP在10～100 mg/L范围,线性关系良好,线性方程为ATP:A=28.924C+14.144,γ=0.999 90;ADP:A=31.504C-12.608,γ=0.999 92;AMP:A=46.539C-5.255,γ=0.999 95。平均回收率为95.3%～103.2%;日内和日间相对标准差(RSD)分别为1.14%～1.75%、2.45%～3.97%。结论高效液相色谱-可变波长检测法可以简便、准确、有效地测定急性重复低氧小鼠脑组织中ATP、ADP和AMP的含量。

High performance liquid chromatographic determination of adenosine phosphates in the mouse brain

Objective To establish a method for the quantitative determination of adenosine phosphates in mouse brain by high performance liquid chromatography-variable wavelength detector(HPLC-VWD). Methods The protein in mouse brain tissue sample was precipitated with perchloric acid. Separation was performed on a reverse phase Supelco Discovery C18 column(150 mm×4.6 mm, 5 μm), equipped with a pre-column of the same material by Agilent 1200 HPLC-VWD. The wavelength was 254 nm. The mobile phase contained methanol-30 mmol/L potassium dihydrogen phosphate buffer, pH 5.85(5∶95, v/v) . The flow rate was set at 1.0 mL/min and the injection volume was 20 μL. The column temperature was 30 ℃. Results The linear relationship between the range(10~100) mg/L was good. The linear equations were ATP: A=28.924C+14.144, γ=0.999 90; ADP: A=31.504C-12.608, γ=0.999 92; AMP: A=46.539C-5.255, γ=0.999 95. The average recovery rate was 95.3%~103.2%. The RSDs of intra-and interday were 1.14%~1.75% and 2.45%~3.97%, respectively. Conclusion The experimental data proved that the HPLC-VWD method could detect the concentration of ATP, ADP and AMP in the mouse brain on repeated hypoxic exposures accurately, rapidly and simply.