在線固相萃取—高效液相色譜法同時(shí)測(cè)定人血清中氯氮平、奎硫平和利培酮的含量

沈廣虎等

摘 要 使用雙梯度液相色譜系統(tǒng)紫外檢測(cè)器，建立了在線固相萃取液相色譜法全自動(dòng)、快速、同時(shí)測(cè)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統(tǒng)，為了提高分離的互補(bǔ)性，使用Capcell MF Ph1柱作為在線固相萃取柱，Acclaim C18柱作為分析柱。在線固相萃取柱以乙腈水體系作為流動(dòng)相，流速1 mL/min梯度洗脫； 分析柱以乙腈100 mmol/L醋酸銨溶液作為流動(dòng)相，流速1 mL/min，梯度洗脫。樣品溶液注入到在線固相萃取苯基柱中，根據(jù)此柱的限進(jìn)機(jī)制，血清中的蛋白等大分子物質(zhì)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；通過(guò)閥切換使用雙梯度液相色譜系統(tǒng)的分析泵將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轉(zhuǎn)移到分析柱中進(jìn)行二次分離，采用外標(biāo)法測(cè)定氯氮平、奎琉平和利培酮的含量，整個(gè)前處理和分析過(guò)程僅需18 min。

3.3 方法線性范圍、檢出限及重現(xiàn)性

按優(yōu)化后的色譜條件，分別測(cè)定了工作溶液，以峰面積對(duì)濃度進(jìn)行線性擬合，氯氮平在10～1800 μg/L范圍內(nèi)線性良好， 線性相關(guān)系數(shù)為0.9996，檢出限為1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范圍內(nèi)線性良好，線性相關(guān)系數(shù)為0.9996，檢出限為0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范圍內(nèi)線性良好，線性相關(guān)系數(shù)為0.9996，檢出限為3.8 μg/L（S/N=3）。取高中低3種濃度的標(biāo)準(zhǔn)溶液連續(xù)進(jìn)樣次，得出峰面積RSD均小于3.8%。

3.4 樣品測(cè)試

取按2.4節(jié)處理過(guò)的血清1 mL，按2.2節(jié)的色譜條件進(jìn)行測(cè)定。每個(gè)樣品均測(cè)定3次，在所測(cè)樣品中均未檢出氯氮平、奎琉平和利培酮。高中低3個(gè)濃度的人血清加標(biāo)回收率如表2所示，氯氮平、奎琉平和利培酮在低濃度時(shí)回收率在100%～119%，中間濃度時(shí)回收率在97%～106%，高濃度時(shí)回收率在98%～106%之間。表明本方法可用于人血清中線性濃度范圍內(nèi)的氯氮平、奎琉平和利培酮的準(zhǔn)確測(cè)定。人血清測(cè)定色譜圖如圖3所示。

本研究通過(guò)閥切換將固相萃取與高效液相色譜相結(jié)合，建立了在線固相萃取方法，生物樣品在高速離心之后直接進(jìn)樣，自動(dòng)實(shí)現(xiàn)蛋白和部分雜質(zhì)的去除以及目標(biāo)化合物的富集，有效降低了干擾，并提高了方法靈敏度。

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 使用雙梯度液相色譜系統(tǒng)紫外檢測(cè)器，建立了在線固相萃取液相色譜法全自動(dòng)、快速、同時(shí)測(cè)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統(tǒng)，為了提高分離的互補(bǔ)性，使用Capcell MF Ph1柱作為在線固相萃取柱，Acclaim C18柱作為分析柱。在線固相萃取柱以乙腈水體系作為流動(dòng)相，流速1 mL/min梯度洗脫； 分析柱以乙腈100 mmol/L醋酸銨溶液作為流動(dòng)相，流速1 mL/min，梯度洗脫。樣品溶液注入到在線固相萃取苯基柱中，根據(jù)此柱的限進(jìn)機(jī)制，血清中的蛋白等大分子物質(zhì)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；通過(guò)閥切換使用雙梯度液相色譜系統(tǒng)的分析泵將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轉(zhuǎn)移到分析柱中進(jìn)行二次分離，采用外標(biāo)法測(cè)定氯氮平、奎琉平和利培酮的含量，整個(gè)前處理和分析過(guò)程僅需18 min。

3.3 方法線性范圍、檢出限及重現(xiàn)性

按優(yōu)化后的色譜條件，分別測(cè)定了工作溶液，以峰面積對(duì)濃度進(jìn)行線性擬合，氯氮平在10～1800 μg/L范圍內(nèi)線性良好， 線性相關(guān)系數(shù)為0.9996，檢出限為1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范圍內(nèi)線性良好，線性相關(guān)系數(shù)為0.9996，檢出限為0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范圍內(nèi)線性良好，線性相關(guān)系數(shù)為0.9996，檢出限為3.8 μg/L（S/N=3）。取高中低3種濃度的標(biāo)準(zhǔn)溶液連續(xù)進(jìn)樣次，得出峰面積RSD均小于3.8%。

3.4 樣品測(cè)試

取按2.4節(jié)處理過(guò)的血清1 mL，按2.2節(jié)的色譜條件進(jìn)行測(cè)定。每個(gè)樣品均測(cè)定3次，在所測(cè)樣品中均未檢出氯氮平、奎琉平和利培酮。高中低3個(gè)濃度的人血清加標(biāo)回收率如表2所示，氯氮平、奎琉平和利培酮在低濃度時(shí)回收率在100%～119%，中間濃度時(shí)回收率在97%～106%，高濃度時(shí)回收率在98%～106%之間。表明本方法可用于人血清中線性濃度范圍內(nèi)的氯氮平、奎琉平和利培酮的準(zhǔn)確測(cè)定。人血清測(cè)定色譜圖如圖3所示。

本研究通過(guò)閥切換將固相萃取與高效液相色譜相結(jié)合，建立了在線固相萃取方法，生物樣品在高速離心之后直接進(jìn)樣，自動(dòng)實(shí)現(xiàn)蛋白和部分雜質(zhì)的去除以及目標(biāo)化合物的富集，有效降低了干擾，并提高了方法靈敏度。

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 使用雙梯度液相色譜系統(tǒng)紫外檢測(cè)器，建立了在線固相萃取液相色譜法全自動(dòng)、快速、同時(shí)測(cè)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統(tǒng)，為了提高分離的互補(bǔ)性，使用Capcell MF Ph1柱作為在線固相萃取柱，Acclaim C18柱作為分析柱。在線固相萃取柱以乙腈水體系作為流動(dòng)相，流速1 mL/min梯度洗脫； 分析柱以乙腈100 mmol/L醋酸銨溶液作為流動(dòng)相，流速1 mL/min，梯度洗脫。樣品溶液注入到在線固相萃取苯基柱中，根據(jù)此柱的限進(jìn)機(jī)制，血清中的蛋白等大分子物質(zhì)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；通過(guò)閥切換使用雙梯度液相色譜系統(tǒng)的分析泵將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轉(zhuǎn)移到分析柱中進(jìn)行二次分離，采用外標(biāo)法測(cè)定氯氮平、奎琉平和利培酮的含量，整個(gè)前處理和分析過(guò)程僅需18 min。

3.3 方法線性范圍、檢出限及重現(xiàn)性

按優(yōu)化后的色譜條件，分別測(cè)定了工作溶液，以峰面積對(duì)濃度進(jìn)行線性擬合，氯氮平在10～1800 μg/L范圍內(nèi)線性良好， 線性相關(guān)系數(shù)為0.9996，檢出限為1.3 μg/L（S/N=3）；奎琉平在3.6～640 μg/L范圍內(nèi)線性良好，線性相關(guān)系數(shù)為0.9996，檢出限為0.93 μg/L（S/N=3）；利培酮在0.71～128 μg/L 范圍內(nèi)線性良好，線性相關(guān)系數(shù)為0.9996，檢出限為3.8 μg/L（S/N=3）。取高中低3種濃度的標(biāo)準(zhǔn)溶液連續(xù)進(jìn)樣次，得出峰面積RSD均小于3.8%。

3.4 樣品測(cè)試

取按2.4節(jié)處理過(guò)的血清1 mL，按2.2節(jié)的色譜條件進(jìn)行測(cè)定。每個(gè)樣品均測(cè)定3次，在所測(cè)樣品中均未檢出氯氮平、奎琉平和利培酮。高中低3個(gè)濃度的人血清加標(biāo)回收率如表2所示，氯氮平、奎琉平和利培酮在低濃度時(shí)回收率在100%～119%，中間濃度時(shí)回收率在97%～106%，高濃度時(shí)回收率在98%～106%之間。表明本方法可用于人血清中線性濃度范圍內(nèi)的氯氮平、奎琉平和利培酮的準(zhǔn)確測(cè)定。人血清測(cè)定色譜圖如圖3所示。

本研究通過(guò)閥切換將固相萃取與高效液相色譜相結(jié)合，建立了在線固相萃取方法，生物樣品在高速離心之后直接進(jìn)樣，自動(dòng)實(shí)現(xiàn)蛋白和部分雜質(zhì)的去除以及目標(biāo)化合物的富集，有效降低了干擾，并提高了方法靈敏度。

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）