A method was developed for simultaneous determination of Clozapine, Quetiapine and Risperidone in human serum by fully automated online solid phase extraction ( SPE )-high performance liquid chromatography. With Capcell MF Ph-1 column as SPE cartridge and Acclaim C18 as analytical column, high selectivity could be achieved by this method according to the selective complementarity of the two columns. In the experiment, ACN-H2 O was used as the SPE mobile phase with a flow rate of 1 mL/min and ACN-10 mmol/L NH4 Ac was used as the analytical mobile phase with a flow rate of 1 mL/min. Serum samples were injected directly into the SPE column and the biological matrix was washed out with the loading solvent. By rotation of the switching valve, Clozapine, Quetiapine and Risperidone were eluted from the SPE cartridge in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase. The whole time of the online SPE purification and chromatographic separation of the analytes was 18 min. Calibration curve of Clozapine with good linearity ( r=0 . 9996 ) was obtained in the range of 10-1800 μg/L in human serum, and the recoveries at low, medium and high concentration levels were 118. 4%, 105. 0% and 105 . 4%. Calibration curve of Quetiapine with good linearity ( r=0 . 9997 ) was obtained in the range of 3 . 6-640 μg/L in human serum, and the recoveries at low, medium and high concentration levels were 112. 8%, 101 . 1% and 101 . 5%. Calibration curve of Risperidone with good linearity ( r=0 . 9995 ) was obtained in the range of 0. 71-128 μg/L in human serum, and the recoveries at low, medium and high concentration levels were 100. 7%, 97. 2% and 98. 8%. In conclusion, the established automated online SPE-HPLC-UV method demonstrated good performance in terms of linearity, specificity, limits of quantification, and was successfully utilized to quantify Clozapine, Quetiapine and Risperidone in human serum.%使用双梯度液相色谱系统紫外检测器,建立了在线固相萃取液相色谱法全自动、快速、同时测定人血清中氯氮平、奎琉平和利培酮的含量。本方法采用了反相系统,为了提高分离的互补性,使用Capcell MF Ph-1柱作为在线固相萃取柱, Acclaim C18柱作为分析柱。在线固相萃取柱以乙腈-水体系作为流动相,流速1 mL/min梯度洗脱；分析柱以乙腈-100 mmol/L醋酸铵溶液作为流动相,流速1 mL/min,梯度洗脱。样品溶液注入到在线固相萃取苯基柱中,根据此柱的限进机制,血清中的蛋白等大分子物质不被保留而排出,而氯氮平、奎琉平和利培酮等小分子化合物得到保留；通过阀切换使用双梯度液相色谱系统的分析泵将固相萃取柱上保留的氯氮平、奎琉平和利培酮等小分子化合物转移到分析柱中进行二次分离,采用外标法测定氯氮平、奎琉平和利培酮的含量,整个前处理和分析过程仅需18 min。氯氮平在10~1800μg/L浓度范围内相关系数为0.9996,低、中、高浓度的平均回收率分别为118.4％,105.0％和105.4％；奎琉平在3.6~640μg/L浓度范围内相关系数r为0.9997,低、中、高浓度的平均回收率分别为112.8％,101.1％和101.5％；利培酮在0.71~128μg/L浓度范围内相关系数为0.9995,低、中、高浓度的平均回收率分别为100.7％,97.2％和98.8％。结果表明,本方法可极大地提高样品分析效率,快速准确测定人血清中氯氮平、奎琉平和利培酮的含量。
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