RP-HPLC法测定全血、血浆和红细胞中氯氮平及2种主要代谢物

RP-HPLC determination of clozapine and its two major metabolites in whole blood,plasma and red blood cells

目的 :建立一种测定全血、血浆和红细胞中氯氮平 (CZP)、N 去甲基氯氮平 (N CZP)和氯氮平N 氧化物 (CZP NO)浓度的RP HPLC法。方法 :标本用乙酸乙酯提取后再经 0 .1mol·L-1盐酸反提取 ,以利培酮为内标 ,流动相为甲醇 乙腈 水 (16 .0∶36 .5∶4 7.5 ) ,色谱柱为Inertsil ODS 3柱 ,柱温为 4 0℃ ,检测波长为 2 5 4nm ,流速为0 .9mL·min-1。结果 :全血、血浆和红细胞中CZP和N CZP的最低检测浓度均为 7μg·L-1;血浆中CZP NO的最低检测浓度为 13μg·L-1,全血和红细胞中为 14 μg·L-1。全血、血浆和红细胞中CZP和N CZP的绝对回收率均为85 %～ 95 % ,血浆中CZP NO的绝对回收率 >82 % ,全血和红细胞中CZP NO的绝对回收率约为 70 %。日内和日间RSD均小于 7%。结论 :本法操作简便 ,灵敏度高 ,重复性好 ,适合于临床检测。

Objective:To establish a RP HPLC method for the simultaneous determination of clozapine(CZP) and its two major metabolites N demethylclozapine( N CZP) and clozapine N oxide (CZP NO) in human whole blood,plasma and red blood cells.Methods:Risperidone was used as internal standard. Blood samples were extracted with ethyl acetate and further purified with 0.1mol·L -1 hydrochloric acid.The compounds were separated on an Inertsil ODS 3 column maintained at the temperature of 40℃and were monitored by ultraviolet detector at 254nm.The mobile phase was methanol acetonitrile water(16.0∶36.5∶47.5) and the flow rate was 0.9ml·min -1 .Results:When 0.5mL blood sample was used for the analysis, the lowest limit of detected concentration was both 7μg·L -1 for CZP and N CZP in human whole bolld,plasma and red blood cells,and 13μg·L -1 for CZP NO in plasma and 14μg·L -1 for CZP NO in human whole blood and red blood cells.The absolute recovery was both 85%～95% for CZP and N CZP in human whole blood,plasma and red blood cells,and over 82% for CZP NO in plasma,and 70% for CZP NO in human whole blood and red blood cells. The within day and between day variance was proved to be less than 7%.Conclusion:This method was validated with respect to simplicity,accuracy, sensitivity and precision.