人血浆中纳美芬和阿扑吗啡测定方法的建立及应用
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摘要
目的建立灵敏、专属、快速的LC／MS／MS法，测定血浆中的纳美芬和阿扑吗啡，并分别用于二者低剂量制剂的临床药动学研究。
方法测定人血浆中纳美芬浓度时，200μL血浆样品经沉淀蛋白处理后，以甲醇-5mmol／L醋酸铵(75：25，v／v)为流动相，经Zorbax XDB C_(18)柱分离，采用API 4000型三重四极杆串联质谱仪电喷雾电离源，正离子方式检测，多反应监测(MRM)，用于定量分析的离子反应分别为m／z340→m／z268 (纳美芬)和m／z286→m／z 185 (内标，氢吗啡酮)。测定人血浆中阿扑吗啡时，200μL血浆样品经液-液萃取处理后，以乙腈-水-甲酸(40：60：0.1，v／v／v)为流动相，采用Zorbax XDB C_8柱分离，采用TSQ Quantum Ultra型三重四极杆串联质谱仪电喷雾电离源，正离子方式检测，选择反应监测(SRM)，用于定量分析的离子反应分别为m／z 268→m／z(237+219)(阿扑吗啡)和m／z 286→m／z 185(内标，氢吗啡酮)。
结果测定纳美芬的线性范围为10.0～5000pg／mL，定量下限为10.0 pg／mL。日内、日间精密度(RSD)小于10.1％，准确度(RE)在-3.4％至1.8％范围内。测定阿扑吗啡的线性范围为0.010～20.0ng／mL，定量下限为0.010 ng／mL。日内、日间精密度(RSD)小于7.9％，准确度(RE)在-1.7％至1.0％范围内。
结论本文所建立的LC／MS／MS法灵敏、专属、快速，可用于纳美芬和阿扑吗啡的血药浓度测定及二者低剂量制剂的临床药物动力学研究。
OBJECTIVE To develop and validate sensitive, rapid and specific liquid chromatographic/tandem mass spectrometric methods for quantitative analyses of nalmefene and apomorphine in human plasma and application to pharmacokinetic studies.
METHODS After a simple one-step protein precipitation with methanol, nalmefene and the internal standard hydromorphone were chromatographed on a Zorbax XDB C_(18) column and detected by MS/MS with a Turbo Ionspray interface on an MDS SCIEX API 4000 tandem mass spectrometer. Multiple reaction monitoring (MRM) adopting the precursor to product ion transitions of m/z 340→m/z 268 and m/z 286→m/z 185 was used to quantify nalmefene and the internal standard, respectively. Apomorphine was first prepared from plasma samples by liquid-liquid extraction, and then separated with the endogenous interferences on a Zorbax XDB C_8 column. The mobile phase consisted of acetonitrile-water-formic acid (40: 60: 0.1, v/v/v) at a flow rate of 0.50 mL/min. A TSQ Quantum Ultra triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, was operated in the positive ion mode. Selective reaction monitoring (SRM) using the precursor to product ion combinations of m/z 268→m/z (237 + 219) and m/z 286→m/z 185 was employed to determine apomorphine and the internal standard hydromorphone, respectively.
RESULTS The linear calibration curve was obtained in the concentration range from 10.0 to 5000 pg/mL for nalmefene. The intra- and inter-run precisions determined from QC samples, expressed as relative standard deviation (RSD), were no more than 6.0% and 10.1%, respectively, while accuracy ranged from -3.4% to 1.8% in terms of relative error (RE). The standard calibration curve for apomorphine was linear in the concentration range from 0.010 to 20.0 ng/mL in human plasma. Determined from QC samples for apomorphine, the intra- and inter-run precisions were less than 5.6% and 7.9%, respectively, and the accuracy was from -1.7% to 1.0%.
CONCLUSION The validated methods were successfully applied to evaluate the clinical pharmacokinetics of low dose of nalmefene and apomorphine.
方法测定人血浆中纳美芬浓度时，200μL血浆样品经沉淀蛋白处理后，以甲醇-5mmol／L醋酸铵(75：25，v／v)为流动相，经Zorbax XDB C_(18)柱分离，采用API 4000型三重四极杆串联质谱仪电喷雾电离源，正离子方式检测，多反应监测(MRM)，用于定量分析的离子反应分别为m／z340→m／z268 (纳美芬)和m／z286→m／z 185 (内标，氢吗啡酮)。测定人血浆中阿扑吗啡时，200μL血浆样品经液-液萃取处理后，以乙腈-水-甲酸(40：60：0.1，v／v／v)为流动相，采用Zorbax XDB C_8柱分离，采用TSQ Quantum Ultra型三重四极杆串联质谱仪电喷雾电离源，正离子方式检测，选择反应监测(SRM)，用于定量分析的离子反应分别为m／z 268→m／z(237+219)(阿扑吗啡)和m／z 286→m／z 185(内标，氢吗啡酮)。
结果测定纳美芬的线性范围为10.0～5000pg／mL，定量下限为10.0 pg／mL。日内、日间精密度(RSD)小于10.1％，准确度(RE)在-3.4％至1.8％范围内。测定阿扑吗啡的线性范围为0.010～20.0ng／mL，定量下限为0.010 ng／mL。日内、日间精密度(RSD)小于7.9％，准确度(RE)在-1.7％至1.0％范围内。
结论本文所建立的LC／MS／MS法灵敏、专属、快速，可用于纳美芬和阿扑吗啡的血药浓度测定及二者低剂量制剂的临床药物动力学研究。
OBJECTIVE To develop and validate sensitive, rapid and specific liquid chromatographic/tandem mass spectrometric methods for quantitative analyses of nalmefene and apomorphine in human plasma and application to pharmacokinetic studies.
METHODS After a simple one-step protein precipitation with methanol, nalmefene and the internal standard hydromorphone were chromatographed on a Zorbax XDB C_(18) column and detected by MS/MS with a Turbo Ionspray interface on an MDS SCIEX API 4000 tandem mass spectrometer. Multiple reaction monitoring (MRM) adopting the precursor to product ion transitions of m/z 340→m/z 268 and m/z 286→m/z 185 was used to quantify nalmefene and the internal standard, respectively. Apomorphine was first prepared from plasma samples by liquid-liquid extraction, and then separated with the endogenous interferences on a Zorbax XDB C_8 column. The mobile phase consisted of acetonitrile-water-formic acid (40: 60: 0.1, v/v/v) at a flow rate of 0.50 mL/min. A TSQ Quantum Ultra triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, was operated in the positive ion mode. Selective reaction monitoring (SRM) using the precursor to product ion combinations of m/z 268→m/z (237 + 219) and m/z 286→m/z 185 was employed to determine apomorphine and the internal standard hydromorphone, respectively.
RESULTS The linear calibration curve was obtained in the concentration range from 10.0 to 5000 pg/mL for nalmefene. The intra- and inter-run precisions determined from QC samples, expressed as relative standard deviation (RSD), were no more than 6.0% and 10.1%, respectively, while accuracy ranged from -3.4% to 1.8% in terms of relative error (RE). The standard calibration curve for apomorphine was linear in the concentration range from 0.010 to 20.0 ng/mL in human plasma. Determined from QC samples for apomorphine, the intra- and inter-run precisions were less than 5.6% and 7.9%, respectively, and the accuracy was from -1.7% to 1.0%.
CONCLUSION The validated methods were successfully applied to evaluate the clinical pharmacokinetics of low dose of nalmefene and apomorphine.
引文
1．梁文权 主编．生物药剂学与药物动力学(第2版)．北京：人民卫生出版社，2003，155-323．
2. Lopea LL, Yu X, Cui D, Davis MR. Research methods & applications: Identification of drug metabolites in biological matrices by intelligent automated liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom, 1998, 12: 1756-1760.
3. Lee MS, Kerns EH. LC/MS applications in drug development. Mass Spectrom Rev, 1999, 18: 187-279.
4. Dixon R, Gentile J, Hsiao J, et al. Nalmefene: Safety and kinetics after single and multiple oral dose of a new opioid antagonist. J Clin Pharmacol, 1987, 27: 233-239.
5. Gonzalez J, Brogden R. Naltrexone: A review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy in the management of opioid dependence. Drugs, 1988, 35: 192-213.
6. Aitkenhead A, Derbyshire D, Pinnock C, et al. Pharmacokinetics of intravenous naloxone in healthy volunteers. Anesthesiology, 1984, 61: A381.
7. Gal T, DiFazio C, Dixon R. Prolonged blockade of opioid effect with oral nalmefene. Clin Pharmacol Ther, 1986, 40: 537-542.
8．任爱国．纳美芬的药理作用及临床应用概况．临床医学，1996，10：66-68．
9. Caldwell RW. Action of the opioid antagonist, nalmefene, and congeners on reperfusion cardiac arrhythmias and regional left coronary blood flow. Pharmacology, 1990, 41: 161-163.
10. Billington CJ, Shafer RB, Morley JE. Effects of opioid blockade with nalmefene in older impotent men. Life Sci, 1990, 47: 799-802.
11. Behrmano DL, Bresnahan JC, Beatie MS. A comparison of YM-14673, U-50488H, and nalmefene after spinal cord injury in the rat. Exp Neurol, 1993, 119: 258-260.
12. Radvanyi T, Marin F, Bikhazi G, et al. Antagonism of the postoperative respiratory depression caused by large doses of morphine. Anesthesiology, 1990, 73: A1172.
13. Chumpa A, Kaplan R, Burns M, et al. Nalmefene for elective reversal of procedural sedation in children. Am J Emerg Med, 2001, 19: 545-548.
14. Kaplan J, Marx J. Effectiveness and safety of intravenous nalmefene for emergency department patients with suspected narcotic overdose: a pilot study. Ann Emerg Med, 1993, 22:187-190.
15. Mason B, Salvato F, Williams L, et al. A double-blind, placebo-controlled study of oral nalmefene for alcohol dependence. Arch Gen Psychiatry, 1999, 56: 719-724.
16. Scan CS et al. Martindale. Great Britain; The Pharmaceutical Press 33 (2002) 1014.
17. Matzke G, Frye R, Alexander A, et al. The effect of renal insufficiency and hemodialysis on the pharmacokinetics of nalmefne. J Clin Pharmacol, 1996, 36:144-151.
18. Heaton J, Morales A, Adams M. Recovery of erectile function by the oral administration of apomorphine. Urology, 1995, 45: 200-206.
19. O'Sullivan J, Hughes A. Apomorphine-induced penile erections in Parkinson's disease. Mov Disord, 1998, 13: 536-539.
20．阿扑吗啡用于勃起功能障碍[J]．德国临床用药，2001，4：42．
21. Cotzias G, Papavasiliou P, Tolosa E, et al. Treatment of Parkinson's disease with apomorphine. N Engl J Med, 1976, 294: 567-572.
22. Gancher S, Nutt J, Woodward W. Absorption of apomorphine by various routes in parkinsonism. Mov Disord, 1991, 6: 212-216.
23. Stibe CM, Kempster PA, Lees AJ, et al. Subcutaneous apomorphine in parkinsonian on-off oscillations. Lancet, 1988, 1:403-406.
24. Van Laar T, Jansen EN, Essink AW, et al. Intranasal apomorphine in parkinsonian on-off fluctuations. Arch Neurol, 1992, 49: 482-484.
25. Scan CS, et al. Martindale. Great Britain; The Pharmaceutical Press 33 (2002) 1164-1165.
26. Danhof M, Van der Geest R, Van Laar T, et al. An integrated pharmacokinetic-pharmacodynamic approach to optimization of R-apomorphine delivery in Parkinson's disease. Adv Drug Deliv Rev, 1998, 33: 253-263.
27. Dixon R, Howes J, Gentile J, et al. Nalmefene: Intravenous safety and kinetics of a new opioid antagonist. Clin Pharmacol Ther, 1986, 39: 49-53.
28. Frye BF, Matzke GR, Schade R, et al. Effects of liver disease on the disposition of the opioid antagonist nalmefene. ClinPharmacol Ther, 1997, 61: 15-23.
29. Chou JZ, Albeck H, Kreek MJ, Determination of nalmefene in plasma by high-performance liquid chromatography with electrochemical detection and its application in pharmacokinetic studies, J Chromatogr, 1993, 613: 359-364.
30. Xie S, Suckow RF, Mason BJ, et al. Rapid and sensitive determination of nalmefene in human plasma by gas chromatography-mass spectrometry, J Chromatogr B, 2002, 773: 143-149.
31. Fang WB, Andrenyak DM, Moody DE, et al. Determination of nalmefene by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, J Anal Toxicol, 2005, 29:169-174.
32. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem, 2003, 75: 3019-3030.
33. Shah VP, Midha KK, Hulse JD, et al. Bioanalytical methods validation — a revisit with a decade of progress. Pharm Res, 2000, 17: 1551-1557.
34．《化学药物临床药代动力学研究技术指导原则》(【H】GCL1-2)[S]．国家食品药品监督管理局．国食药监注 2005．
35. Arglolas A, Hedlund H..The pharmacology and clinical pharmacokinetics of apomorphine SL. BJU International, 2001, 88 (Suppl): 18-21.
36. Ingram WM, Priston MJ, Sewell GJ. Improved assay for R(-)-apomorphine with application to clinical pharrnacokinetic studies in Parkinson's disease, J Chromatogr B, 2006, 831: 1-7.
37. Libert F, Coudoré F, Richard D, et al. Development of a gas chromatographic/mass spectrometric method to quantify R-apomorphine, R-apocodeine and R- norapomorphine in human plasma and urine. J Mass Spectrom, 2005, 40: 1521-1525.
38. Van der Geest R, Kruger P, Gubbens-Stibbe JM, et al. Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease. J Chromatogr B, 1997, 702:131-141.
39. Yang B, Zhu JB, Deng CH, et al. Development of a sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of apomorphine in canine plasma. Rapid Commun Mass Spectrom, 2006, 20: 1883-1888.
40．郭继芬，张爱军，赵玲 等．液相色谱-串联质谱法测定人血浆中阿扑吗啡．分析测试学报，2004，23：39．
41. Sam E, Augustijns P, Verbeke N. Stability of apomorphine in plasma and its determination by high-performance liquid chromatography with electrochemical detection. J Chromatogr B, 1994, 658: 311-317.
2. Lopea LL, Yu X, Cui D, Davis MR. Research methods & applications: Identification of drug metabolites in biological matrices by intelligent automated liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom, 1998, 12: 1756-1760.
3. Lee MS, Kerns EH. LC/MS applications in drug development. Mass Spectrom Rev, 1999, 18: 187-279.
4. Dixon R, Gentile J, Hsiao J, et al. Nalmefene: Safety and kinetics after single and multiple oral dose of a new opioid antagonist. J Clin Pharmacol, 1987, 27: 233-239.
5. Gonzalez J, Brogden R. Naltrexone: A review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy in the management of opioid dependence. Drugs, 1988, 35: 192-213.
6. Aitkenhead A, Derbyshire D, Pinnock C, et al. Pharmacokinetics of intravenous naloxone in healthy volunteers. Anesthesiology, 1984, 61: A381.
7. Gal T, DiFazio C, Dixon R. Prolonged blockade of opioid effect with oral nalmefene. Clin Pharmacol Ther, 1986, 40: 537-542.
8．任爱国．纳美芬的药理作用及临床应用概况．临床医学，1996，10：66-68．
9. Caldwell RW. Action of the opioid antagonist, nalmefene, and congeners on reperfusion cardiac arrhythmias and regional left coronary blood flow. Pharmacology, 1990, 41: 161-163.
10. Billington CJ, Shafer RB, Morley JE. Effects of opioid blockade with nalmefene in older impotent men. Life Sci, 1990, 47: 799-802.
11. Behrmano DL, Bresnahan JC, Beatie MS. A comparison of YM-14673, U-50488H, and nalmefene after spinal cord injury in the rat. Exp Neurol, 1993, 119: 258-260.
12. Radvanyi T, Marin F, Bikhazi G, et al. Antagonism of the postoperative respiratory depression caused by large doses of morphine. Anesthesiology, 1990, 73: A1172.
13. Chumpa A, Kaplan R, Burns M, et al. Nalmefene for elective reversal of procedural sedation in children. Am J Emerg Med, 2001, 19: 545-548.
14. Kaplan J, Marx J. Effectiveness and safety of intravenous nalmefene for emergency department patients with suspected narcotic overdose: a pilot study. Ann Emerg Med, 1993, 22:187-190.
15. Mason B, Salvato F, Williams L, et al. A double-blind, placebo-controlled study of oral nalmefene for alcohol dependence. Arch Gen Psychiatry, 1999, 56: 719-724.
16. Scan CS et al. Martindale. Great Britain; The Pharmaceutical Press 33 (2002) 1014.
17. Matzke G, Frye R, Alexander A, et al. The effect of renal insufficiency and hemodialysis on the pharmacokinetics of nalmefne. J Clin Pharmacol, 1996, 36:144-151.
18. Heaton J, Morales A, Adams M. Recovery of erectile function by the oral administration of apomorphine. Urology, 1995, 45: 200-206.
19. O'Sullivan J, Hughes A. Apomorphine-induced penile erections in Parkinson's disease. Mov Disord, 1998, 13: 536-539.
20．阿扑吗啡用于勃起功能障碍[J]．德国临床用药，2001，4：42．
21. Cotzias G, Papavasiliou P, Tolosa E, et al. Treatment of Parkinson's disease with apomorphine. N Engl J Med, 1976, 294: 567-572.
22. Gancher S, Nutt J, Woodward W. Absorption of apomorphine by various routes in parkinsonism. Mov Disord, 1991, 6: 212-216.
23. Stibe CM, Kempster PA, Lees AJ, et al. Subcutaneous apomorphine in parkinsonian on-off oscillations. Lancet, 1988, 1:403-406.
24. Van Laar T, Jansen EN, Essink AW, et al. Intranasal apomorphine in parkinsonian on-off fluctuations. Arch Neurol, 1992, 49: 482-484.
25. Scan CS, et al. Martindale. Great Britain; The Pharmaceutical Press 33 (2002) 1164-1165.
26. Danhof M, Van der Geest R, Van Laar T, et al. An integrated pharmacokinetic-pharmacodynamic approach to optimization of R-apomorphine delivery in Parkinson's disease. Adv Drug Deliv Rev, 1998, 33: 253-263.
27. Dixon R, Howes J, Gentile J, et al. Nalmefene: Intravenous safety and kinetics of a new opioid antagonist. Clin Pharmacol Ther, 1986, 39: 49-53.
28. Frye BF, Matzke GR, Schade R, et al. Effects of liver disease on the disposition of the opioid antagonist nalmefene. ClinPharmacol Ther, 1997, 61: 15-23.
29. Chou JZ, Albeck H, Kreek MJ, Determination of nalmefene in plasma by high-performance liquid chromatography with electrochemical detection and its application in pharmacokinetic studies, J Chromatogr, 1993, 613: 359-364.
30. Xie S, Suckow RF, Mason BJ, et al. Rapid and sensitive determination of nalmefene in human plasma by gas chromatography-mass spectrometry, J Chromatogr B, 2002, 773: 143-149.
31. Fang WB, Andrenyak DM, Moody DE, et al. Determination of nalmefene by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, J Anal Toxicol, 2005, 29:169-174.
32. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem, 2003, 75: 3019-3030.
33. Shah VP, Midha KK, Hulse JD, et al. Bioanalytical methods validation — a revisit with a decade of progress. Pharm Res, 2000, 17: 1551-1557.
34．《化学药物临床药代动力学研究技术指导原则》(【H】GCL1-2)[S]．国家食品药品监督管理局．国食药监注 2005．
35. Arglolas A, Hedlund H..The pharmacology and clinical pharmacokinetics of apomorphine SL. BJU International, 2001, 88 (Suppl): 18-21.
36. Ingram WM, Priston MJ, Sewell GJ. Improved assay for R(-)-apomorphine with application to clinical pharrnacokinetic studies in Parkinson's disease, J Chromatogr B, 2006, 831: 1-7.
37. Libert F, Coudoré F, Richard D, et al. Development of a gas chromatographic/mass spectrometric method to quantify R-apomorphine, R-apocodeine and R- norapomorphine in human plasma and urine. J Mass Spectrom, 2005, 40: 1521-1525.
38. Van der Geest R, Kruger P, Gubbens-Stibbe JM, et al. Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease. J Chromatogr B, 1997, 702:131-141.
39. Yang B, Zhu JB, Deng CH, et al. Development of a sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of apomorphine in canine plasma. Rapid Commun Mass Spectrom, 2006, 20: 1883-1888.
40．郭继芬，张爱军，赵玲 等．液相色谱-串联质谱法测定人血浆中阿扑吗啡．分析测试学报，2004，23：39．
41. Sam E, Augustijns P, Verbeke N. Stability of apomorphine in plasma and its determination by high-performance liquid chromatography with electrochemical detection. J Chromatogr B, 1994, 658: 311-317.