LC/MS/MS法测定血浆中两种他汀类降血脂药及应用
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摘要
目的：拟建立两种专属、灵敏的LC／MS／MS法，分别测定人血浆中瑞舒伐他汀浓度和辛伐他汀浓度，并应用于瑞舒伐他汀临床药代动力学和辛伐他汀人体生物利用度及等效性研究。
方法：测定人血浆中瑞舒伐他汀的浓度时，以氢氯噻嗪为内标，血浆样品经乙醚液—液萃取后，以甲醇-水(75∶25 v／v，1％氨水调pH至6)为流动相，采用Zorbax XDB C_(18)柱进行分离，通过(-)ESI源离子化，以多反应监测(MRM)方式进行检测。用于定量分析的离子反应分别为m／z 480→m／z 418(瑞舒伐他汀)和m／z 296→m／z 269(氢氯噻嗪)。测定人血浆中辛伐他汀时，200μL血浆样品经乙醚—二氯甲烷(3∶2，v／v)液—液萃取后，以乙腈-水-甲酸(55∶45∶0.25，v／v／v)为流动相，采用Zorbax SB C_(18)短柱分离，通过(+)ESI离子化，以选择反应监测(SRM)方式进行检测。用于定量分析的离子反应分别为m／z 441→m／z 325(辛伐他汀)和m／z 427→m／z 325(洛伐他汀)。
结果：测定瑞舒伐他汀的线性范围为0.020-60.0 ng／mL，定量下限为0.020 ng／mL，日内、日间精密度(RSD％)小于8.5％，准确度(RE％)在-0.3～1.9％范围内。测定辛伐他汀的线性范围为0.05-100ng／mL，定量下限为0.05 ng／mL。日内、日间精密度(RSD％)小于7.9％，准确度(RE％)在-2.2～-0.6％范围内。已成功应用于瑞舒伐他汀和辛伐他汀的血浆浓度测定及临床药动学和人体生物利用度及等效性研究。
结论：所建立的LC／MS／MS法专属、灵敏，可成功用于瑞舒伐他汀和辛伐他汀的血浆浓度测定以及临床药动学研究。
Objective: Two rapid, sensitive and specific methods for quantitative analyses of two statins drugs in plasma will be developed and validated by liquid chromatography/tandem mass spectrometry in this thesis. The methods will be applied to pharmacokinetic studies.
Method: Determination of rosuvastatin in human plasma by liquid chromatography-tandem mass spectrometry. Samples spiked with the analyte and internal standard, hydrochlorothiazide, were processed using liquid-liquid extraction. The extract was chromatographed on a Zorbax XDB C_(18) column. The mobile phase consisted of methanol-water (75: 25, v/v, adjusted to pH 6 by 1% ammonia aqueous), at a flow rate of 0.50 mL/min. An API4000 tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring (MRM) using the precursor product ion combinations of m/z 480→m/z 418 and m/z 296→m/z 269 were used to quantify rosuvastatin and hydrochlorothiazide, respectively. After simple liquid-liquid extraction, simvastatin and the internal standard lovastatin were chromatographed on a Zorbax SB C_(18) column and detected by MS/MS with an electrospray ionization interface in a Thermo Finnigan TSQ Quantum Ultra tandem mass spectrometer. Selected reaction monitoring (SRM) using the precursor product ion combination of m/z 441→m/z 325 and m/z 427→m/z 325 was used to quantify simvastatin and the internal standard, respectively.
Results: The linear calibration curves were obtained in the concentration range of 0.020-60.0 ng/mL for rosuvastatin. The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was less than 8.5%. The accuracy was within -0.3 to 1.9%. The standard calibration curve for simvastatin was linear in the concentration range of 0.05-100 ng/mL in human plasma. The intra- and inter-run precisions were less than 7.9%, determined from QC samples for simvastain, and accuracy was within -2.2 to -0.6% in terms of relative error.
Conclusion: The methods were successfully applied for the evaluation of the pharmacokinetics and bioequivalence ofrosuvastatin and simvastatin.
方法：测定人血浆中瑞舒伐他汀的浓度时，以氢氯噻嗪为内标，血浆样品经乙醚液—液萃取后，以甲醇-水(75∶25 v／v，1％氨水调pH至6)为流动相，采用Zorbax XDB C_(18)柱进行分离，通过(-)ESI源离子化，以多反应监测(MRM)方式进行检测。用于定量分析的离子反应分别为m／z 480→m／z 418(瑞舒伐他汀)和m／z 296→m／z 269(氢氯噻嗪)。测定人血浆中辛伐他汀时，200μL血浆样品经乙醚—二氯甲烷(3∶2，v／v)液—液萃取后，以乙腈-水-甲酸(55∶45∶0.25，v／v／v)为流动相，采用Zorbax SB C_(18)短柱分离，通过(+)ESI离子化，以选择反应监测(SRM)方式进行检测。用于定量分析的离子反应分别为m／z 441→m／z 325(辛伐他汀)和m／z 427→m／z 325(洛伐他汀)。
结果：测定瑞舒伐他汀的线性范围为0.020-60.0 ng／mL，定量下限为0.020 ng／mL，日内、日间精密度(RSD％)小于8.5％，准确度(RE％)在-0.3～1.9％范围内。测定辛伐他汀的线性范围为0.05-100ng／mL，定量下限为0.05 ng／mL。日内、日间精密度(RSD％)小于7.9％，准确度(RE％)在-2.2～-0.6％范围内。已成功应用于瑞舒伐他汀和辛伐他汀的血浆浓度测定及临床药动学和人体生物利用度及等效性研究。
结论：所建立的LC／MS／MS法专属、灵敏，可成功用于瑞舒伐他汀和辛伐他汀的血浆浓度测定以及临床药动学研究。
Objective: Two rapid, sensitive and specific methods for quantitative analyses of two statins drugs in plasma will be developed and validated by liquid chromatography/tandem mass spectrometry in this thesis. The methods will be applied to pharmacokinetic studies.
Method: Determination of rosuvastatin in human plasma by liquid chromatography-tandem mass spectrometry. Samples spiked with the analyte and internal standard, hydrochlorothiazide, were processed using liquid-liquid extraction. The extract was chromatographed on a Zorbax XDB C_(18) column. The mobile phase consisted of methanol-water (75: 25, v/v, adjusted to pH 6 by 1% ammonia aqueous), at a flow rate of 0.50 mL/min. An API4000 tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring (MRM) using the precursor product ion combinations of m/z 480→m/z 418 and m/z 296→m/z 269 were used to quantify rosuvastatin and hydrochlorothiazide, respectively. After simple liquid-liquid extraction, simvastatin and the internal standard lovastatin were chromatographed on a Zorbax SB C_(18) column and detected by MS/MS with an electrospray ionization interface in a Thermo Finnigan TSQ Quantum Ultra tandem mass spectrometer. Selected reaction monitoring (SRM) using the precursor product ion combination of m/z 441→m/z 325 and m/z 427→m/z 325 was used to quantify simvastatin and the internal standard, respectively.
Results: The linear calibration curves were obtained in the concentration range of 0.020-60.0 ng/mL for rosuvastatin. The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was less than 8.5%. The accuracy was within -0.3 to 1.9%. The standard calibration curve for simvastatin was linear in the concentration range of 0.05-100 ng/mL in human plasma. The intra- and inter-run precisions were less than 7.9%, determined from QC samples for simvastain, and accuracy was within -2.2 to -0.6% in terms of relative error.
Conclusion: The methods were successfully applied for the evaluation of the pharmacokinetics and bioequivalence ofrosuvastatin and simvastatin.
引文
1．张骁，束梅英，张韬。他汀类降血脂药物的研究进展及市场前景(一)．中国医药情报，2003，9：35-39．
2. Rutishauser J. The role of statins in clinical medicine-LDL-cholestrol lowering and beyond. Swiss Med Wkly. 2006, 136: 41-49.
3. Piechowski-Jozwiak B, Bogousslavsky J. Antihypertensive and lipid lowering treatment in stroke prevention: current state and future. Acta Neurol Belg. 2005, 105: 57-61.
4. Thavendiranathan P, Bagai A, Brookhart MA, et al. Primary prevention of cardiovascular diseases with statin therapy: a meta-analysis of randomized controlled trials. Arch Intern Med. 2006, 166: 2307-2313.
5. Mauro VF. Clinical pharmacokinetics and practical applications of simvastatin. Clin Pharmacokinet. 1993, 24: 195-202.
6. Lennernas H, Fager G. Pharmacodynamics and pharmacokinetics of the HMG-CoA reductase inhibitors. Similarities and differences. Clin Pharmacokinet. 1997, 32: 403-425.
7. Culhane NS, Lettieri SL, Skae JR, et al. Rosuvastatin for the treatment of hypercholesterolemia. Pharmacotherapy. 2005, 25: 990-1000.
8. Nezasa K, Higaki K, Matsumura T, Inazawa K, Hasegawa H, Nakano M, Koike M. Liver-specific distribution of rosuvastatin in rats: comparison with pravastatin and simvastatin. Drug Metab.Dispos. 2002, 30: 1158-1163.
9. Martin PD, Mitchell PD, Schneck DW. Pharmacodynamic effects and pharmacokinetics of a new HMG-CoA reductase inhibitor, rosuvastatin, after moring or evening administration in healthy volunteers. Br J Clin Pharmacol. 2002, 54: 472-477.
10．戴德哉主编．临床药理学．北京：中国医药科技出版社，2000，28．
11．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．
12. Lim Ck, Lord G. Current developments in LC-MS for pharmaceutical analysis. Biol Pharm Bull. 2002, 25: 547-557.
13. 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.
14. Martin PD, Warwick MJ, Dane AL, et al. A double-blind, randomized, incomplete crossover trial to assess the dose proportionality of rosuvastatin in healthy volunteers. Clin Ther, 2003; 25: 2215-2224.
15. Kumer TR, Shitut NR, Kumer PK, et al. Determination of rosuvastatin in rat plasma by HPLC: validation and its application to pharmacokinetic studies. Biomed Chromatogr. 2006; 20: 881.
16. Vittal S, Shitut NR, Kumar TR, et al. Simultaneous quantitation of rosuvastatin and gemfibrozil in human plasma by high-performance liquid chromatography and its application to a pharmacokinetic study. Biomed. Chromatogr. 2006; 20: 1252.
17. Hull CK, Penman AD, Smith CK, et al. Quantification of rosuvastatin in human plasma by automated solid-phase extration using tandem mass spectrometric detection. J Chromatogr B, 2002; 772: 219-228.
18. Oudhoff KA, Sangster T, Thomas E, et al. Application of microbore HPLC in combination with tandem MS for the quantification of rosuvastatin in human plasma. J Chromatogr B, 2006; 832: 191-196.
19. Trivedi RK, Kallem RR, Mullangi R, et al, Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC-MS/MS with electrospray ionization: Assay development, validation and application to a clinical study. J Pharm Biomed Anal, 2005; 39: 661-669.
20. Xu DH, Ruan ZH, Zhou Q, et al. Quantitative determination of rosuvastatin in human plasma by liquid chromatography with electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2006; 20: 2369.
21. Antignac JP, Wasch KD, Monteau F, et al. The ion suppression phenomenon in liquid chromatography-mass spectrometry and its consequences in the field of residue analysis. Anal Chim Acta. 2005; 529: 129-136.
22．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．
23. Karnes HT, March C. Precision, accuracy and data acceptance criteria in biopharmaceutical analysis. Pharm Res. 1993, 10: 1420-1422.
24. Shah VP, Midha KK, Hulse JD, et al. Bioanalytical methods validation-a revisit with a decade of progress. Pharm Res. 2000, 17: 1551-1557.
25．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】GCL1-2)．国食药监注[2005]106号文件．2005，3．
26．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．
27. Lee E, Ryan E, Birmingham B, et al. Rosuvastatin pharmacokinetics and pharmacogenetics in white and Asian subjects residing in the same environment. Clin Pharmacol Ther. 2005, 78: 330-341.
28. Kantola T, Kivisto KT, Neuvonen PJ. Erythromycin and verapamil considerably increase serum simvastatin and simvastatin acid concentrations. Clin Pharmacol Ther. 1998, 64: 177-182.
29. Vickers S, Duncan CA, Chen IW, et al. Metabolic disposition studies on simvastatin, a cholesterol-lowering prodrug. Drug Metab Dispos. 1990, 18: 138-145.
30. Morris MJ, Gilbert JD, Hsieh JY, et al. Determination of the HMG-CoA reductase inhibitors simvastatin, lovastatin, and pravastatin in plasma by gas chromatography/chemical ionization mass spectrometry. Biol Mass Spectrom. 1993, 22:1-8.
31. Wang L, Asgharnejad M. Second-derivative UV spectrometric determination of simvastatin in its tablet dosage form. J Pharm Boimed. Anal, 2000, 21: 1243-1248.
32. Carlucci G, Mazzeo P, Biordi L, et al. Simultaneous determination of simvastatin and its hydroxy acid form in human plasma by high-performance liquid chromatography with UV detection. J Pharm Boimed Anal. 1992, 10: 693-697.
33. Ochiai H, Uchiyama N, Imagaki K, et al. Determination of simvastatin and its active metabolite in human plasma by column-switching high-performance liquid chromatography with fluorescence detection after derivatization with 1-bromoacetylpyrene. J Chromatogr B. 1997, 694: 211-217.
34. Zhao JJ, Xie IH, Yang AY, et al. Quantitation of simvastatin and its β-hydroxy acid in human plasma by liquid-liquid cartridge extraction and liquid chromatography/tandem mass spectrometry. J Mass Spectrom. 2000, 35: 1133-1143.
35. Jemal M, Ouyang Z, Powell M. Direct-injection LC-MS-MS method for high-throughput simultaneous quantitation of simvastatin and simvastatin acid in human plasma. J Pharm Biomed Anal, 2000, 23: 323-340.
36. Yang HT, Feng Y, Luan YW. Determination of simvastain in human plasma by liquid chromatography-mass spectrometry. J Chromatogr B. 2003, 78: 369-375.
37. Yang AY, Sun L, Musson DG, et al. Application of a novel ultra-low elution volume 96-well solid-phase extraction method to the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma. J Pharm Biomed Anal. 2005, 38: 521-527.
38. Barrett B, HuclováJ, Borek-Dohalsky V, et al. Validated HPLC-MS/MS method for simultaneous determination of simvastatin and simvastatin hydroxy acid in human plasma. J Pharm Biomed Anal. 2006, 41: 517-526.
2. Rutishauser J. The role of statins in clinical medicine-LDL-cholestrol lowering and beyond. Swiss Med Wkly. 2006, 136: 41-49.
3. Piechowski-Jozwiak B, Bogousslavsky J. Antihypertensive and lipid lowering treatment in stroke prevention: current state and future. Acta Neurol Belg. 2005, 105: 57-61.
4. Thavendiranathan P, Bagai A, Brookhart MA, et al. Primary prevention of cardiovascular diseases with statin therapy: a meta-analysis of randomized controlled trials. Arch Intern Med. 2006, 166: 2307-2313.
5. Mauro VF. Clinical pharmacokinetics and practical applications of simvastatin. Clin Pharmacokinet. 1993, 24: 195-202.
6. Lennernas H, Fager G. Pharmacodynamics and pharmacokinetics of the HMG-CoA reductase inhibitors. Similarities and differences. Clin Pharmacokinet. 1997, 32: 403-425.
7. Culhane NS, Lettieri SL, Skae JR, et al. Rosuvastatin for the treatment of hypercholesterolemia. Pharmacotherapy. 2005, 25: 990-1000.
8. Nezasa K, Higaki K, Matsumura T, Inazawa K, Hasegawa H, Nakano M, Koike M. Liver-specific distribution of rosuvastatin in rats: comparison with pravastatin and simvastatin. Drug Metab.Dispos. 2002, 30: 1158-1163.
9. Martin PD, Mitchell PD, Schneck DW. Pharmacodynamic effects and pharmacokinetics of a new HMG-CoA reductase inhibitor, rosuvastatin, after moring or evening administration in healthy volunteers. Br J Clin Pharmacol. 2002, 54: 472-477.
10．戴德哉主编．临床药理学．北京：中国医药科技出版社，2000，28．
11．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．
12. Lim Ck, Lord G. Current developments in LC-MS for pharmaceutical analysis. Biol Pharm Bull. 2002, 25: 547-557.
13. 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.
14. Martin PD, Warwick MJ, Dane AL, et al. A double-blind, randomized, incomplete crossover trial to assess the dose proportionality of rosuvastatin in healthy volunteers. Clin Ther, 2003; 25: 2215-2224.
15. Kumer TR, Shitut NR, Kumer PK, et al. Determination of rosuvastatin in rat plasma by HPLC: validation and its application to pharmacokinetic studies. Biomed Chromatogr. 2006; 20: 881.
16. Vittal S, Shitut NR, Kumar TR, et al. Simultaneous quantitation of rosuvastatin and gemfibrozil in human plasma by high-performance liquid chromatography and its application to a pharmacokinetic study. Biomed. Chromatogr. 2006; 20: 1252.
17. Hull CK, Penman AD, Smith CK, et al. Quantification of rosuvastatin in human plasma by automated solid-phase extration using tandem mass spectrometric detection. J Chromatogr B, 2002; 772: 219-228.
18. Oudhoff KA, Sangster T, Thomas E, et al. Application of microbore HPLC in combination with tandem MS for the quantification of rosuvastatin in human plasma. J Chromatogr B, 2006; 832: 191-196.
19. Trivedi RK, Kallem RR, Mullangi R, et al, Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC-MS/MS with electrospray ionization: Assay development, validation and application to a clinical study. J Pharm Biomed Anal, 2005; 39: 661-669.
20. Xu DH, Ruan ZH, Zhou Q, et al. Quantitative determination of rosuvastatin in human plasma by liquid chromatography with electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2006; 20: 2369.
21. Antignac JP, Wasch KD, Monteau F, et al. The ion suppression phenomenon in liquid chromatography-mass spectrometry and its consequences in the field of residue analysis. Anal Chim Acta. 2005; 529: 129-136.
22．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．
23. Karnes HT, March C. Precision, accuracy and data acceptance criteria in biopharmaceutical analysis. Pharm Res. 1993, 10: 1420-1422.
24. Shah VP, Midha KK, Hulse JD, et al. Bioanalytical methods validation-a revisit with a decade of progress. Pharm Res. 2000, 17: 1551-1557.
25．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】GCL1-2)．国食药监注[2005]106号文件．2005，3．
26．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．
27. Lee E, Ryan E, Birmingham B, et al. Rosuvastatin pharmacokinetics and pharmacogenetics in white and Asian subjects residing in the same environment. Clin Pharmacol Ther. 2005, 78: 330-341.
28. Kantola T, Kivisto KT, Neuvonen PJ. Erythromycin and verapamil considerably increase serum simvastatin and simvastatin acid concentrations. Clin Pharmacol Ther. 1998, 64: 177-182.
29. Vickers S, Duncan CA, Chen IW, et al. Metabolic disposition studies on simvastatin, a cholesterol-lowering prodrug. Drug Metab Dispos. 1990, 18: 138-145.
30. Morris MJ, Gilbert JD, Hsieh JY, et al. Determination of the HMG-CoA reductase inhibitors simvastatin, lovastatin, and pravastatin in plasma by gas chromatography/chemical ionization mass spectrometry. Biol Mass Spectrom. 1993, 22:1-8.
31. Wang L, Asgharnejad M. Second-derivative UV spectrometric determination of simvastatin in its tablet dosage form. J Pharm Boimed. Anal, 2000, 21: 1243-1248.
32. Carlucci G, Mazzeo P, Biordi L, et al. Simultaneous determination of simvastatin and its hydroxy acid form in human plasma by high-performance liquid chromatography with UV detection. J Pharm Boimed Anal. 1992, 10: 693-697.
33. Ochiai H, Uchiyama N, Imagaki K, et al. Determination of simvastatin and its active metabolite in human plasma by column-switching high-performance liquid chromatography with fluorescence detection after derivatization with 1-bromoacetylpyrene. J Chromatogr B. 1997, 694: 211-217.
34. Zhao JJ, Xie IH, Yang AY, et al. Quantitation of simvastatin and its β-hydroxy acid in human plasma by liquid-liquid cartridge extraction and liquid chromatography/tandem mass spectrometry. J Mass Spectrom. 2000, 35: 1133-1143.
35. Jemal M, Ouyang Z, Powell M. Direct-injection LC-MS-MS method for high-throughput simultaneous quantitation of simvastatin and simvastatin acid in human plasma. J Pharm Biomed Anal, 2000, 23: 323-340.
36. Yang HT, Feng Y, Luan YW. Determination of simvastain in human plasma by liquid chromatography-mass spectrometry. J Chromatogr B. 2003, 78: 369-375.
37. Yang AY, Sun L, Musson DG, et al. Application of a novel ultra-low elution volume 96-well solid-phase extraction method to the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma. J Pharm Biomed Anal. 2005, 38: 521-527.
38. Barrett B, HuclováJ, Borek-Dohalsky V, et al. Validated HPLC-MS/MS method for simultaneous determination of simvastatin and simvastatin hydroxy acid in human plasma. J Pharm Biomed Anal. 2006, 41: 517-526.