血浆中氟桂利嗪和HV-08的定量分析方法研究
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摘要
本文有两部分组成。
一、液相色谱-串联质谱法测定血浆中的氟桂利嗪
目的：建立专属、灵敏的液相色谱-串联质谱法测定人血浆中氟桂利嗪的浓度。方法：0.25 mL血浆样品经液-液萃取后，以乙腈-水-甲酸(80∶20∶0.5，v／v／v)为流动相，Zorbax SB C_(18)柱分离，通过大气压化学电离源四极杆串联质谱，以选择反应监测方式进行检测。用于定量分析的离子反应分别为m／z 405→m／z 203(氟桂利嗪)和m／z 256→m／z 167(内标，苯海拉明)。结果：测定血浆中氟桂利嗪方法的线性范围为0.2～200 ng／mL，定量下限为0.2ng／mL。日内、日间精密度(RSD)均小于6.5％，准确度(RE)在±0.9％以内。结论：该方法选择性强，灵敏度高，适用于氟桂利嗪临床药动学研究。
二、血浆中HV-08的定量分析方法研究
目的：建立快速、灵敏的液相色谱-串联质谱法测定血浆中的HV-08，研究HV-08在Wistar大鼠和Beagle犬体内的药物动力学性质。方法：血浆样品经沉淀蛋白提取，以苯海拉明为内标，流动相为甲醇-水-甲酸(60∶40∶0.2，v／v／v)，采用Diamonsil C_(18)柱进行色谱分离，通过大气压化学电离源四极杆串联质谱，以选择反应监测方式进行检测。用于定量分析的离子反应分别为m／z 563→m／z 58 (HV-08)和m／z 256→m／z 167(内标，苯海拉明)。结果：该法用于测定HV-08的线性范围为0.75～1000 ng／mL，定量下限为0.75 ng／mL。应用本法对Wistar大鼠和Beagle犬分别静脉注射和三个剂量组灌胃给予HV-08后的药物动力学进行研究。结论：该方法选择性强，灵敏度高，适用于临床前药动学研究。
Two rapid, sensitive and specific LC/MS/MS methods for quantitative analyses of flunarizine and HV-08 in plasma were developed and validated.1. Determination of flunarizine in plasma by LC/MS/MSObjective To develop a sensitive and specific LC/MS/MS method for determination of flunarizine in human plasma. Methods Flunarizine and internal standard diphenhydramine were extracted from 0.25 mL plasma using liquid-liquid extraction, then separated on a Zorbax SB C_(18) column. The mobile phase consisted of acetonitrile-water-formic acid (80: 20: 0.5, v/v/v). A Finnigan TSQ tandem mass spectrometer equipped with atmospheric pressure chemical ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring mode using the precursor to product ion of m/z 405 to m/z 203 and m/z 256 to m/z 167 was performed to quantify flunarizine and the internal standard respectively. Results The linear calibration curves were obtained in the concentration range of 0.2-200 ng/mL. The lower limit of quantification was 0.2 ng/mL. The inter- and intra-day precision (RSD) was below 6.5 %, and the accuracy (RE) was within±0.9 % calculated from QC samples. Conclusion The method was proved to be specific, sensitive, and suitable for clinical investigation of flunarizine pharmacokinetics.2. Quantitative analyses of HV-08 in plasma by LC/MS/MSObjective To develop a rapid, sensitive and highly selective liquid chromatography-tandem mass spectrometry method for determination of HV-08 in plasma. Methods The analytes were extracted from plasma samples by protein precipitation, separated on a Diamonsil C_(18) column and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization source. The mobile phase consisted of methanol-water-formic acid (60:40:0.2, v/v/v). Diphenhydramine was used as the internal standard. Selected reaction monitoring using the precursor→product ion combinations of m/z 563→m/z 58 and m/z 256→m/z 167 was used to quantify HV-08 and internal standard, respectively. Results The method has a lower limit of quantification (LLOQ) of 0.75 ng/mL for HV-08. The linear calibration curves were obtained in the concentration range of 0.75-1000 ng/mL. The method was applied to study the pharmacokinetics of HV-08 in rats and dogs after oral and i.v. injection administration of HV-08. Conclusion The method was proved to be specific, sensitive, and suitable for investigation of HV-08 pharmacokinetics in rats and dogs.
一、液相色谱-串联质谱法测定血浆中的氟桂利嗪
目的：建立专属、灵敏的液相色谱-串联质谱法测定人血浆中氟桂利嗪的浓度。方法：0.25 mL血浆样品经液-液萃取后，以乙腈-水-甲酸(80∶20∶0.5，v／v／v)为流动相，Zorbax SB C_(18)柱分离，通过大气压化学电离源四极杆串联质谱，以选择反应监测方式进行检测。用于定量分析的离子反应分别为m／z 405→m／z 203(氟桂利嗪)和m／z 256→m／z 167(内标，苯海拉明)。结果：测定血浆中氟桂利嗪方法的线性范围为0.2～200 ng／mL，定量下限为0.2ng／mL。日内、日间精密度(RSD)均小于6.5％，准确度(RE)在±0.9％以内。结论：该方法选择性强，灵敏度高，适用于氟桂利嗪临床药动学研究。
二、血浆中HV-08的定量分析方法研究
目的：建立快速、灵敏的液相色谱-串联质谱法测定血浆中的HV-08，研究HV-08在Wistar大鼠和Beagle犬体内的药物动力学性质。方法：血浆样品经沉淀蛋白提取，以苯海拉明为内标，流动相为甲醇-水-甲酸(60∶40∶0.2，v／v／v)，采用Diamonsil C_(18)柱进行色谱分离，通过大气压化学电离源四极杆串联质谱，以选择反应监测方式进行检测。用于定量分析的离子反应分别为m／z 563→m／z 58 (HV-08)和m／z 256→m／z 167(内标，苯海拉明)。结果：该法用于测定HV-08的线性范围为0.75～1000 ng／mL，定量下限为0.75 ng／mL。应用本法对Wistar大鼠和Beagle犬分别静脉注射和三个剂量组灌胃给予HV-08后的药物动力学进行研究。结论：该方法选择性强，灵敏度高，适用于临床前药动学研究。
Two rapid, sensitive and specific LC/MS/MS methods for quantitative analyses of flunarizine and HV-08 in plasma were developed and validated.1. Determination of flunarizine in plasma by LC/MS/MSObjective To develop a sensitive and specific LC/MS/MS method for determination of flunarizine in human plasma. Methods Flunarizine and internal standard diphenhydramine were extracted from 0.25 mL plasma using liquid-liquid extraction, then separated on a Zorbax SB C_(18) column. The mobile phase consisted of acetonitrile-water-formic acid (80: 20: 0.5, v/v/v). A Finnigan TSQ tandem mass spectrometer equipped with atmospheric pressure chemical ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring mode using the precursor to product ion of m/z 405 to m/z 203 and m/z 256 to m/z 167 was performed to quantify flunarizine and the internal standard respectively. Results The linear calibration curves were obtained in the concentration range of 0.2-200 ng/mL. The lower limit of quantification was 0.2 ng/mL. The inter- and intra-day precision (RSD) was below 6.5 %, and the accuracy (RE) was within±0.9 % calculated from QC samples. Conclusion The method was proved to be specific, sensitive, and suitable for clinical investigation of flunarizine pharmacokinetics.2. Quantitative analyses of HV-08 in plasma by LC/MS/MSObjective To develop a rapid, sensitive and highly selective liquid chromatography-tandem mass spectrometry method for determination of HV-08 in plasma. Methods The analytes were extracted from plasma samples by protein precipitation, separated on a Diamonsil C_(18) column and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization source. The mobile phase consisted of methanol-water-formic acid (60:40:0.2, v/v/v). Diphenhydramine was used as the internal standard. Selected reaction monitoring using the precursor→product ion combinations of m/z 563→m/z 58 and m/z 256→m/z 167 was used to quantify HV-08 and internal standard, respectively. Results The method has a lower limit of quantification (LLOQ) of 0.75 ng/mL for HV-08. The linear calibration curves were obtained in the concentration range of 0.75-1000 ng/mL. The method was applied to study the pharmacokinetics of HV-08 in rats and dogs after oral and i.v. injection administration of HV-08. Conclusion The method was proved to be specific, sensitive, and suitable for investigation of HV-08 pharmacokinetics in rats and dogs.
引文
1．吴镭主编．药学科学前沿与发展方向．北京：中国医药科技出版社，2000：54-59．
2．凌树森主编．治疗药物监测新理论与新方法．北京：中国医药科技出版社，2002，4：290-292．
3．李好枝主编．体内药物分析．北京：中国医药科技出版社，2003，7：141-328．
4．蒋学华主编．药物评价方法概论．成都：四川大学出版社，2005：240-241．
5．张君仁，臧恒昌主编．体內药物分析．北京：化学工业出版社，2002，9：11-12．
6. Battle KD and Myers P. History of gas chromatography. Trends in analytical chemistry. 2002, 21: 547-557.
7．张玉奎，张维冰，邹汉法主编．分析化学手册．第六分册：液相色谱分析．第二版．北京：化学工业出版社，2000．12：76-86．
8．施奈德LR，格莱吉克 JK，柯克兰JJ．实用高效液相色谱法的建立．中文版．北京：科学出版社，2000，5．
9. Matsuo T, Seyama Y. Introduction to modem biological mass spectrometry. J Mass Spectrom. 2000, 35: 114-130.
10. Ermer J, Vogel M. Application of hyphenated LC-MS techniques in pharmaceutical analysis. Biomed Chromatogr. 2000, 14: 373-383.
11. Oliveira EJ, Watson DG. Liquid chromatography-mass spectrometry in the study of the metabolism of drugs and other xenobiotics. Biomed Chromatogr. 2000, 14: 351-372.
12. Niessen WM. Progress in liquid chromatography-mass spectrometry instrumentation and its impact on high throughput screening. J Chromatogr A. 2003, 1000: 413-436.
13. Lim Ck, Lord G. Current developments in LC-MS for pharmaceutical analysis. Biol Pharm Bull. 2002, 25: 547-557.
14. Plumb RS, Dear GJ, Mallett DN, et al. Quantitative analysis of pharmaceuticals in biological fluids using high-performance liquid chromatography coupled to mass spectrometry: a review. Xenobiotica. 2001, 31: 599-617.
15. Geoghegan KF and Kelly MA. Biochemical applications of mass spectrometry in pharmaceutical drug discovery. Mass Spetrom Rev. 2005, 24: 347-366.
16. Weigl M, Tenze G, Steinlechner, et al. A systematic review of currently available pharmacological neuroprotective agents as a sole intervention before anticipated or induced cardiac arrest. Resuscitation. 2005, 65: 21-39.
17. So HS, Park C, Kim HJ, et al. Protective effect of T-type calcium channel blocker flunadzine on cisplatin-induced death of anditory cells. Hearing Research. 2004, 204: 127-139.
18. Visudtibhan A, Lusawat A, Chiemchanya S, et al. Flunarizine for prophylactic treatment of childhood migraine. J Med Assoc Thai. 2004, 87: 1466-1470.
19．舒青，毕宏伟．氟桂利嗪的药理研究与临床应用进展．中国药师．2000，3：146-147．
20. Kazusaburo K, Nobuhiro K, Masako O, et al. Determination of flunarizine in serum by high performance liquid chromatography and its clinical application. Yakugaku Zasshi. 1988, 108: 226-231.
21. Nieder M and Jaeger H. High-performance liquid chromatographic assay of flunarizine in plasma and its application to biopharmaceutic investigations. J Chromatogr. 1986, 380: 443-449.
22. Torchin CD, Kapetanovic IM, Yonekawa WD, et al. High-performance liquid chromatographic assay of flunarizine, (E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine, in plasma of epileptic patients. J Chromatogr. 1988, 426: 444-448.
23. Albani F, Riva R, Casucci G, et al. Liquid chromatographic determination of flunarizine in human plasma. J Chromatogr. 1986, 374: 196-199.
24. Woestenborghs R, Michielsen L, Lorreyne W, et al. Sensitive gas chromatographic method for the determination of cinnatizine and flunarizine in biological samples. J Chromatogr. 1982, 232: 85-91.
25. Kapetanovic IM, Torchin CD, Yonekawa WD, et al. Automated capillary gas chromatographic determination of flunarizine. J Chromatogr. 1986, 383: 223-228.
26. Camposflor S. Determination of the calcium antagonist flunarizine in biological fluids by gas-liquid chromatography. J Chromatogr. 1983, 272: 315-323.
27. Fuh M R, Hsieh C J. Determination of flunarizine in rat brain by liquid chromatography-electrospray mass spectrometry. J Chromatogr B. 1999, 736: 167-173.
28．钟大放．以加权最小二乘法建立生物分析标准曲线的若干问题．药物分析杂志．1996，16：343-346．
29. Karnes HT, March C. Precision, accuracy and data acceptance criteria in biopharmaceutical analysis. Pharm Res. 1993, 10: 1420-1422.
30. Shah VP, Midha KK, Hulse JD, et al. Bioanalytical methods validation-a revisit with a decade of progress. Pharm Res. 2000, 17: 1551-1557.
31．国家食品药品监督管理局．化学药物临床药代动力学研究技术指导原则(【H】GCLl-2)．国食药监注[2005]106号文件．2005，3．
32．国家药典委员会编．药物制剂人体生物利用度和等效性试验指导原则．见：中华人民共和国药典，2005年版二部，附录173-176．
33．何海霞，周远大，姚高琼，等．人血浆中盐酸氟桂利嗪的HPLC测定及药代动力学研究．药物分析杂志．2000，20：157-159．
34．刘玉旺，孙培红，赵霞．氟桂利嗪胶囊剂的人体生物等效性研究．中国临床药理学杂志．2001．17：277-280．
35．陈庆宪，罗大林，周远大．盐酸氟桂利嗪的人体药动学及相对生物利用度．华西药学杂志．2000，15：273-275．
36．张会杰，张红，熊玉卿．盐酸氟桂利嗪血药浓度HPLC测定和生物等效性研究．中国临床药理学与治疗学．2003，8：451-453．
37．国家食品药品监督管理局．化学药物非临床药代动力学研究技术指导原则(【H】GPT5-1)．国食药监注[2005]106号文件．2005，3．
2．凌树森主编．治疗药物监测新理论与新方法．北京：中国医药科技出版社，2002，4：290-292．
3．李好枝主编．体内药物分析．北京：中国医药科技出版社，2003，7：141-328．
4．蒋学华主编．药物评价方法概论．成都：四川大学出版社，2005：240-241．
5．张君仁，臧恒昌主编．体內药物分析．北京：化学工业出版社，2002，9：11-12．
6. Battle KD and Myers P. History of gas chromatography. Trends in analytical chemistry. 2002, 21: 547-557.
7．张玉奎，张维冰，邹汉法主编．分析化学手册．第六分册：液相色谱分析．第二版．北京：化学工业出版社，2000．12：76-86．
8．施奈德LR，格莱吉克 JK，柯克兰JJ．实用高效液相色谱法的建立．中文版．北京：科学出版社，2000，5．
9. Matsuo T, Seyama Y. Introduction to modem biological mass spectrometry. J Mass Spectrom. 2000, 35: 114-130.
10. Ermer J, Vogel M. Application of hyphenated LC-MS techniques in pharmaceutical analysis. Biomed Chromatogr. 2000, 14: 373-383.
11. Oliveira EJ, Watson DG. Liquid chromatography-mass spectrometry in the study of the metabolism of drugs and other xenobiotics. Biomed Chromatogr. 2000, 14: 351-372.
12. Niessen WM. Progress in liquid chromatography-mass spectrometry instrumentation and its impact on high throughput screening. J Chromatogr A. 2003, 1000: 413-436.
13. Lim Ck, Lord G. Current developments in LC-MS for pharmaceutical analysis. Biol Pharm Bull. 2002, 25: 547-557.
14. Plumb RS, Dear GJ, Mallett DN, et al. Quantitative analysis of pharmaceuticals in biological fluids using high-performance liquid chromatography coupled to mass spectrometry: a review. Xenobiotica. 2001, 31: 599-617.
15. Geoghegan KF and Kelly MA. Biochemical applications of mass spectrometry in pharmaceutical drug discovery. Mass Spetrom Rev. 2005, 24: 347-366.
16. Weigl M, Tenze G, Steinlechner, et al. A systematic review of currently available pharmacological neuroprotective agents as a sole intervention before anticipated or induced cardiac arrest. Resuscitation. 2005, 65: 21-39.
17. So HS, Park C, Kim HJ, et al. Protective effect of T-type calcium channel blocker flunadzine on cisplatin-induced death of anditory cells. Hearing Research. 2004, 204: 127-139.
18. Visudtibhan A, Lusawat A, Chiemchanya S, et al. Flunarizine for prophylactic treatment of childhood migraine. J Med Assoc Thai. 2004, 87: 1466-1470.
19．舒青，毕宏伟．氟桂利嗪的药理研究与临床应用进展．中国药师．2000，3：146-147．
20. Kazusaburo K, Nobuhiro K, Masako O, et al. Determination of flunarizine in serum by high performance liquid chromatography and its clinical application. Yakugaku Zasshi. 1988, 108: 226-231.
21. Nieder M and Jaeger H. High-performance liquid chromatographic assay of flunarizine in plasma and its application to biopharmaceutic investigations. J Chromatogr. 1986, 380: 443-449.
22. Torchin CD, Kapetanovic IM, Yonekawa WD, et al. High-performance liquid chromatographic assay of flunarizine, (E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine, in plasma of epileptic patients. J Chromatogr. 1988, 426: 444-448.
23. Albani F, Riva R, Casucci G, et al. Liquid chromatographic determination of flunarizine in human plasma. J Chromatogr. 1986, 374: 196-199.
24. Woestenborghs R, Michielsen L, Lorreyne W, et al. Sensitive gas chromatographic method for the determination of cinnatizine and flunarizine in biological samples. J Chromatogr. 1982, 232: 85-91.
25. Kapetanovic IM, Torchin CD, Yonekawa WD, et al. Automated capillary gas chromatographic determination of flunarizine. J Chromatogr. 1986, 383: 223-228.
26. Camposflor S. Determination of the calcium antagonist flunarizine in biological fluids by gas-liquid chromatography. J Chromatogr. 1983, 272: 315-323.
27. Fuh M R, Hsieh C J. Determination of flunarizine in rat brain by liquid chromatography-electrospray mass spectrometry. J Chromatogr B. 1999, 736: 167-173.
28．钟大放．以加权最小二乘法建立生物分析标准曲线的若干问题．药物分析杂志．1996，16：343-346．
29. Karnes HT, March C. Precision, accuracy and data acceptance criteria in biopharmaceutical analysis. Pharm Res. 1993, 10: 1420-1422.
30. Shah VP, Midha KK, Hulse JD, et al. Bioanalytical methods validation-a revisit with a decade of progress. Pharm Res. 2000, 17: 1551-1557.
31．国家食品药品监督管理局．化学药物临床药代动力学研究技术指导原则(【H】GCLl-2)．国食药监注[2005]106号文件．2005，3．
32．国家药典委员会编．药物制剂人体生物利用度和等效性试验指导原则．见：中华人民共和国药典，2005年版二部，附录173-176．
33．何海霞，周远大，姚高琼，等．人血浆中盐酸氟桂利嗪的HPLC测定及药代动力学研究．药物分析杂志．2000，20：157-159．
34．刘玉旺，孙培红，赵霞．氟桂利嗪胶囊剂的人体生物等效性研究．中国临床药理学杂志．2001．17：277-280．
35．陈庆宪，罗大林，周远大．盐酸氟桂利嗪的人体药动学及相对生物利用度．华西药学杂志．2000，15：273-275．
36．张会杰，张红，熊玉卿．盐酸氟桂利嗪血药浓度HPLC测定和生物等效性研究．中国临床药理学与治疗学．2003，8：451-453．
37．国家食品药品监督管理局．化学药物非临床药代动力学研究技术指导原则(【H】GPT5-1)．国食药监注[2005]106号文件．2005，3．