HPLC法同时测定大鼠肝微粒中原儿茶酸和香草酸的含量

目的：建立测定大鼠离体肝微粒中儿茶酚氧甲基转移酶（ COMT）底物（原儿茶酸）和代谢产物（香草酸）含量的HPLC法。方法离体肝微粒体样品经酸化后，乙酸乙酯提取。以绿原酸作为内标，色谱柱为 Welchrom C18，流动相为甲醇：0.1％磷酸＝25：75，流速为1 ml/min，检测波长为260 nm、326 nm，柱温30℃，进样量20μl。结果原儿茶酸和香草酸分别在0.5-20μg/ml和0.375-15μg/ml范围内线性关系良好，r≥0.9995；原儿茶酸的日内精密度和日间精密度RSD≤13.2％，方法回收率为90.3％-115.4％，提取率≥94.9％；香草酸的日内精密度和日间精密度RSD≤8.5％，方法回收率为93.4％-103.1％，提取回收率≥88.5％。结论该方法灵敏、简便、准确，可用于大鼠肝微体中COMT活性的测定。

Simultaneous determination of content of protocatechuic acid and vanillic acid in rat liver microsomes by HPLC

Objective To establish a HPLC method for the simultaneous determination of content of catechol-Omethyltransferase（ COMT）substrate（ protocatechuic acid）and product（ vanillic acid）in isolated rat liver microsomes. Methods The isolated liver microsome samples were acidified by HCl and then extracted with ethyl acetate;chlorogenic acid was chosen as internal standards;the detection was performed on a Welchrom C18 column,with mobile phase of methanol：0. 1% phosphoric acid=25：75;the flow rate was 1 ml/min and the detection wavelength were set at 260 nm and 326 nm;the column temperature was 30 ℃,and injection volume was 20 μl. Results The linear ranges of protocatechuic acid and vanillic acid were 0. 5-20 μg/ml and 0. 375-15 μg/ml, respectively;the intra-and inter-assay precisions of protocatechuic acid was less than 13. 2%,the method recovery was between 90. 3%and 115. 4%,and the extraction recovery was greater than 94. 9%;while the intra-and inter-assay precisions of vanillic acid were less than 8. 5%,the method recovery was between 93. 4% and 103. 1%,and the extraction recovery was greater than 88. 5%. Conclusions This method is sensitive,simple and accurate and can be applied to detect COMT enzyme activity in isolated rat liver microsomes.