HPLC法测定大鼠肝微粒体中UGT酶的底物

Determination of UGT Substrates in Rats' Liver Microsomes by HPLC

目的 建立测定大鼠肝微粒中UGT酶底物的HPLC方法.方法 选择对硝基酚、对乙酰氨基酚作为UGT的特异性底物.采用高效液相色谱法,分别以非那西丁、咖啡因作为内标,色谱柱为Welchrom C18,体积流量为1 mL/min,柱温为30℃.对硝基酚和对乙酰氨基酚的流动相分别为乙腈-0.01 mol/L的醋酸铵缓冲液（pH5.00）=35∶65和乙腈-水=14∶86,对应的检测波长分别为316 nm和244 nm.结果 对硝基酚和对乙酰氨基酚在0.5～100 μmol/L范围内,线性关系良好.对硝基酚的日内精密度和日间精密度RSD％＜14.7％,方法回收率为89.9％～111.0％,提取回收率＞86.5％.对乙酰氨基酚的日内精密度和日间精密度RSD％＜13.0％,方法回收率105.6％～114.2％,提取回收率＞78.3％.结论 两组方法灵敏、简便、准确,可用于大鼠肝微粒体中UGT酶活性的测定.

Objective To establish a HPLC method for the determination of UGT substrates in isolated rat liver microsomes. Methods P-nitrophenol and acetaminophen were chosen as UGT substrates, with phenacetin and caffeine as internal standards, respectively. The determination was performed on a Welchrom C18 column, with mobile phase of p-nitrophenol was acetonitrile-0. 01mol/L ammonium acetate （pH 5. 00 = 35 ： 65, and for acetaminophen was acetonitrile -water= 14 ： 86. The flow rate was 1 mL/min. Detection wavelength of p- nitrophenol and acetaminophen were set at 316 nm and 244 nm. The column temperature was 30℃, and injection volume was 20 μL. Results The linear ranges of p-nitrophenol and acetaminophen were 0. 5-100μmol/L. The intra-and inter-assay precisions of p-nitrophenol were less than 14.7%, the assay recoveries were between 89.9% and 111.0%, and the extract recoveries were greater than 86. 5%. For acetaminophen, the intra-and inter-assay precisions were less than 13. 0%, the method recoveries were between 105. 6% and 114. 2%, and extraction recovery was greater than 78.3%. Conclusion The two methods were sensitive, simple and accurate, which can be applied for determination of UGT enzyme activity in isolated rat liver microsome.