在线固相萃取—高效液相色谱法同时测定人血清中氯氮平、奎硫平和利培酮的含量
沈广虎等
摘 要 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 Ph1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。
3.3 方法线性范围、检出限及重现性
按优化后的色谱条件，分别测定了工作溶液，以峰面积对浓度进行线性拟合，氯氮平在10～1800 μg/L范围内线性良好， 线性相关系数为0.9996，检出限为1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范围内线性良好，线性相关系数为0.9996，检出限为0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范围内线性良好，线性相关系数为0.9996，检出限为3.8 μg/L（S/N=3）。取高中低3种浓度的标准溶液连续进样次，得出峰面积RSD均小于3.8%。
3.4 样品测试
取按2.4节处理过的血清1 mL，按2.2节的色谱条件进行测定。每个样品均测定3次，在所测样品中均未检出氯氮平、奎琉平和利培酮。高中低3个浓度的人血清加标回收率如表2所示，氯氮平、奎琉平和利培酮在低浓度时回收率在100%～119%，中间浓度时回收率在97%～106%，高浓度时回收率在98%～106%之间。表明本方法可用于人血清中线性浓度范围内的氯氮平、奎琉平和利培酮的准确测定。人血清测定色谱图如图3所示。
本研究通过阀切换将固相萃取与高效液相色谱相结合，建立了在线固相萃取方法，生物样品在高速离心之后直接进样，自动实现蛋白和部分杂质的去除以及目标化合物的富集，有效降低了干扰，并提高了方法灵敏度。
Simultaneous Determination of Clozapine， Quetiapine and
Risperidone in Human Serum with Online Solid Phase
ExtractionHigh Performance Liquid Chromatography
SHEN GuangHu1， GUO LiPing2， LIU LiSheng1， ZHANG WenYing1， LIU XiaoDa*2
1（Tianjin Anding Hospital， Tianjin 300222， China）
2（Thermo Fisher Scientific， Beijing 100080， China）
Abstract 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 Ph1 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， ACNH2O was used as the SPE mobile phase with a flow rate of 1 mL/min and ACN10 mmol/L NH4Ac 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 backflush 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 SPEHPLCUV 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.
Keywords High performance liquid chromatography； Online solid phase extraction； Clozapine； Quetiapine； Risperidone； Serum
（Received 18 July 2014； accepted 14 October 2014）
摘 要 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 Ph1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。
3.3 方法线性范围、检出限及重现性
按优化后的色谱条件，分别测定了工作溶液，以峰面积对浓度进行线性拟合，氯氮平在10～1800 μg/L范围内线性良好， 线性相关系数为0.9996，检出限为1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范围内线性良好，线性相关系数为0.9996，检出限为0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范围内线性良好，线性相关系数为0.9996，检出限为3.8 μg/L（S/N=3）。取高中低3种浓度的标准溶液连续进样次，得出峰面积RSD均小于3.8%。
3.4 样品测试
取按2.4节处理过的血清1 mL，按2.2节的色谱条件进行测定。每个样品均测定3次，在所测样品中均未检出氯氮平、奎琉平和利培酮。高中低3个浓度的人血清加标回收率如表2所示，氯氮平、奎琉平和利培酮在低浓度时回收率在100%～119%，中间浓度时回收率在97%～106%，高浓度时回收率在98%～106%之间。表明本方法可用于人血清中线性浓度范围内的氯氮平、奎琉平和利培酮的准确测定。人血清测定色谱图如图3所示。
本研究通过阀切换将固相萃取与高效液相色谱相结合，建立了在线固相萃取方法，生物样品在高速离心之后直接进样，自动实现蛋白和部分杂质的去除以及目标化合物的富集，有效降低了干扰，并提高了方法灵敏度。
Simultaneous Determination of Clozapine， Quetiapine and
Risperidone in Human Serum with Online Solid Phase
ExtractionHigh Performance Liquid Chromatography
SHEN GuangHu1， GUO LiPing2， LIU LiSheng1， ZHANG WenYing1， LIU XiaoDa*2
1（Tianjin Anding Hospital， Tianjin 300222， China）
2（Thermo Fisher Scientific， Beijing 100080， China）
Abstract 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 Ph1 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， ACNH2O was used as the SPE mobile phase with a flow rate of 1 mL/min and ACN10 mmol/L NH4Ac 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 backflush 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 SPEHPLCUV 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.
Keywords High performance liquid chromatography； Online solid phase extraction； Clozapine； Quetiapine； Risperidone； Serum
（Received 18 July 2014； accepted 14 October 2014）
摘 要 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 Ph1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。
3.3 方法线性范围、检出限及重现性
按优化后的色谱条件，分别测定了工作溶液，以峰面积对浓度进行线性拟合，氯氮平在10～1800 μg/L范围内线性良好， 线性相关系数为0.9996，检出限为1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范围内线性良好，线性相关系数为0.9996，检出限为0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范围内线性良好，线性相关系数为0.9996，检出限为3.8 μg/L（S/N=3）。取高中低3种浓度的标准溶液连续进样次，得出峰面积RSD均小于3.8%。
3.4 样品测试
取按2.4节处理过的血清1 mL，按2.2节的色谱条件进行测定。每个样品均测定3次，在所测样品中均未检出氯氮平、奎琉平和利培酮。高中低3个浓度的人血清加标回收率如表2所示，氯氮平、奎琉平和利培酮在低浓度时回收率在100%～119%，中间浓度时回收率在97%～106%，高浓度时回收率在98%～106%之间。表明本方法可用于人血清中线性浓度范围内的氯氮平、奎琉平和利培酮的准确测定。人血清测定色谱图如图3所示。
本研究通过阀切换将固相萃取与高效液相色谱相结合，建立了在线固相萃取方法，生物样品在高速离心之后直接进样，自动实现蛋白和部分杂质的去除以及目标化合物的富集，有效降低了干扰，并提高了方法灵敏度。
Simultaneous Determination of Clozapine， Quetiapine and
Risperidone in Human Serum with Online Solid Phase
ExtractionHigh Performance Liquid Chromatography
SHEN GuangHu1， GUO LiPing2， LIU LiSheng1， ZHANG WenYing1， LIU XiaoDa*2
1（Tianjin Anding Hospital， Tianjin 300222， China）
2（Thermo Fisher Scientific， Beijing 100080， China）
Abstract 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 Ph1 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， ACNH2O was used as the SPE mobile phase with a flow rate of 1 mL/min and ACN10 mmol/L NH4Ac 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 backflush 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 SPEHPLCUV 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.
Keywords High performance liquid chromatography； Online solid phase extraction； Clozapine； Quetiapine； Risperidone； Serum
（Received 18 July 2014； accepted 14 October 2014）
摘 要 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 Ph1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。
3.3 方法线性范围、检出限及重现性
按优化后的色谱条件，分别测定了工作溶液，以峰面积对浓度进行线性拟合，氯氮平在10～1800 μg/L范围内线性良好， 线性相关系数为0.9996，检出限为1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范围内线性良好，线性相关系数为0.9996，检出限为0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范围内线性良好，线性相关系数为0.9996，检出限为3.8 μg/L（S/N=3）。取高中低3种浓度的标准溶液连续进样次，得出峰面积RSD均小于3.8%。
3.4 样品测试
取按2.4节处理过的血清1 mL，按2.2节的色谱条件进行测定。每个样品均测定3次，在所测样品中均未检出氯氮平、奎琉平和利培酮。高中低3个浓度的人血清加标回收率如表2所示，氯氮平、奎琉平和利培酮在低浓度时回收率在100%～119%，中间浓度时回收率在97%～106%，高浓度时回收率在98%～106%之间。表明本方法可用于人血清中线性浓度范围内的氯氮平、奎琉平和利培酮的准确测定。人血清测定色谱图如图3所示。
本研究通过阀切换将固相萃取与高效液相色谱相结合，建立了在线固相萃取方法，生物样品在高速离心之后直接进样，自动实现蛋白和部分杂质的去除以及目标化合物的富集，有效降低了干扰，并提高了方法灵敏度。
Simultaneous Determination of Clozapine， Quetiapine and
Risperidone in Human Serum with Online Solid Phase
ExtractionHigh Performance Liquid Chromatography
SHEN GuangHu1， GUO LiPing2， LIU LiSheng1， ZHANG WenYing1， LIU XiaoDa*2
1（Tianjin Anding Hospital， Tianjin 300222， China）
2（Thermo Fisher Scientific， Beijing 100080， China）
Abstract 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 Ph1 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， ACNH2O was used as the SPE mobile phase with a flow rate of 1 mL/min and ACN10 mmol/L NH4Ac 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 backflush 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 SPEHPLCUV 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.
Keywords High performance liquid chromatography； Online solid phase extraction； Clozapine； Quetiapine； Risperidone； Serum
（Received 18 July 2014； accepted 14 October 2014）
摘 要 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 Ph1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。
3.3 方法线性范围、检出限及重现性
按优化后的色谱条件，分别测定了工作溶液，以峰面积对浓度进行线性拟合，氯氮平在10～1800 μg/L范围内线性良好， 线性相关系数为0.9996，检出限为1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范围内线性良好，线性相关系数为0.9996，检出限为0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范围内线性良好，线性相关系数为0.9996，检出限为3.8 μg/L（S/N=3）。取高中低3种浓度的标准溶液连续进样次，得出峰面积RSD均小于3.8%。
3.4 样品测试
取按2.4节处理过的血清1 mL，按2.2节的色谱条件进行测定。每个样品均测定3次，在所测样品中均未检出氯氮平、奎琉平和利培酮。高中低3个浓度的人血清加标回收率如表2所示，氯氮平、奎琉平和利培酮在低浓度时回收率在100%～119%，中间浓度时回收率在97%～106%，高浓度时回收率在98%～106%之间。表明本方法可用于人血清中线性浓度范围内的氯氮平、奎琉平和利培酮的准确测定。人血清测定色谱图如图3所示。
本研究通过阀切换将固相萃取与高效液相色谱相结合，建立了在线固相萃取方法，生物样品在高速离心之后直接进样，自动实现蛋白和部分杂质的去除以及目标化合物的富集，有效降低了干扰，并提高了方法灵敏度。
Simultaneous Determination of Clozapine， Quetiapine and
Risperidone in Human Serum with Online Solid Phase
ExtractionHigh Performance Liquid Chromatography
SHEN GuangHu1， GUO LiPing2， LIU LiSheng1， ZHANG WenYing1， LIU XiaoDa*2
1（Tianjin Anding Hospital， Tianjin 300222， China）
2（Thermo Fisher Scientific， Beijing 100080， China）
Abstract 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 Ph1 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， ACNH2O was used as the SPE mobile phase with a flow rate of 1 mL/min and ACN10 mmol/L NH4Ac 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 backflush 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 SPEHPLCUV 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.
Keywords High performance liquid chromatography； Online solid phase extraction； Clozapine； Quetiapine； Risperidone； Serum
（Received 18 July 2014； accepted 14 October 2014）
摘 要 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 Ph1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。
3.3 方法线性范围、检出限及重现性
按优化后的色谱条件，分别测定了工作溶液，以峰面积对浓度进行线性拟合，氯氮平在10～1800 μg/L范围内线性良好， 线性相关系数为0.9996，检出限为1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范围内线性良好，线性相关系数为0.9996，检出限为0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范围内线性良好，线性相关系数为0.9996，检出限为3.8 μg/L（S/N=3）。取高中低3种浓度的标准溶液连续进样次，得出峰面积RSD均小于3.8%。
3.4 样品测试
取按2.4节处理过的血清1 mL，按2.2节的色谱条件进行测定。每个样品均测定3次，在所测样品中均未检出氯氮平、奎琉平和利培酮。高中低3个浓度的人血清加标回收率如表2所示，氯氮平、奎琉平和利培酮在低浓度时回收率在100%～119%，中间浓度时回收率在97%～106%，高浓度时回收率在98%～106%之间。表明本方法可用于人血清中线性浓度范围内的氯氮平、奎琉平和利培酮的准确测定。人血清测定色谱图如图3所示。
本研究通过阀切换将固相萃取与高效液相色谱相结合，建立了在线固相萃取方法，生物样品在高速离心之后直接进样，自动实现蛋白和部分杂质的去除以及目标化合物的富集，有效降低了干扰，并提高了方法灵敏度。
Simultaneous Determination of Clozapine， Quetiapine and
Risperidone in Human Serum with Online Solid Phase
ExtractionHigh Performance Liquid Chromatography
SHEN GuangHu1， GUO LiPing2， LIU LiSheng1， ZHANG WenYing1， LIU XiaoDa*2
1（Tianjin Anding Hospital， Tianjin 300222， China）
2（Thermo Fisher Scientific， Beijing 100080， China）
Abstract 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 Ph1 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， ACNH2O was used as the SPE mobile phase with a flow rate of 1 mL/min and ACN10 mmol/L NH4Ac 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 backflush 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 SPEHPLCUV 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.
Keywords High performance liquid chromatography； Online solid phase extraction； Clozapine； Quetiapine； Risperidone； Serum
（Received 18 July 2014； accepted 14 October 2014）
