摘要
目的：分别建立测定人血浆中的缬沙坦和米氮平的快速、灵敏的液相色谱-串联质谱法，并应用于药物动力学研究以及制剂人体生物利用度和生物等效性研究。方法：50μL含缬沙坦的血浆样品加入内标坎地沙坦，经液—液萃取处理后，以甲醇-5mmol/L乙酸铵(80：20，v/v)为流动相，采用Zorbax SB-C_(18)柱分离；50μL含米氮平的血浆样品加入内标苯海拉明，经沉淀蛋白处理后，以乙腈-水-甲酸(80：20：0.2，v/v/v)为流动相，采用Zorbax SB-C_8柱分离。分别通过电喷雾离子化四极杆串联质谱，以选择反应监测(SRM)方式进行正离子检测，用于监测定量的离子反应分别为m/z436→m/z207，235，291(缬沙坦)和m/z 441→m/z 263(内标)；m/z 266→m/z 195(米氮平)和m/z 256→m/z 167(内标)。药动学参数采用非室模型计算。结果：缬沙坦和米氮平测定方法的线性范围分别为4.0～8000ng/mL和0.18～144.0ng/mL。日内、日间精密度均小于15％，准确度在±15％以内。每个样品测试时间均少于3.6min。应用上述方法研究比较了健康受试者分别单剂量口服缬沙坦160mg以及米氮平30mg后的药物动力学特点，分别进行了两种药物不同制剂的人体生物利用度和生物等效性研究。结论：本文所建立的两种定量方法选择性强，灵敏度高，操作简便，血浆用量少，分别适用于缬沙坦以及米氮平制剂的生物等效性评价及临床药动学研究。
Objective: To develop two sensitive and specific LC/MS/MS methods for respective determination of valsartan and mirtazapine in human plasma and to study the bioequivalences of different formulations containing valsartan and mirtazapine and clinical investigations of their pharmacokinetics, respectively. Method: For determination of the plasma concentration of valsartan, an aliquot of 50-μL plasma was treated by liquid-liquid extraction, then the analytes of interest were analyzed on a Zorbax SB-C_(18) column with the mobile phase consisted of methanol-5 mmol/L ammonium acetate (80: 20, v/v). For determination of the plasma concentration of mirtazapine, an aliquot of 50-μL plasma was treated by precipitation. The analytes of interest were separated on a Zorbax SB-C_8 column with the mobile phase consisting of acetonitrile-water-formic acid (80: 20: 0.2, v/v/v). A Thermo Finnigan TSQ Ultra tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor → product ion combination of m/z 436 → m/z 207,235,291 and m/z 441→m/z 263 was used to respectively quantify valsartan and candesartan, m/z 266 → m/z 195 and m/z 256→ m/z 167 was used to respectively quantify mirtazapine and diphenhydramine. Results: The linear calibration curves were obtained in the concentration range of 4.0-8000 ng/mL and 0.18-144.0 ng/mL for valsartan and mirtazapine, respectively. The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was less than 15%. Accuracy determined at three QC concentrations was within ± 15% as terms of relative error (RE). Each plasma sample was chromatographed within 3.6 min. Conclusion: The methods were sensitive and convenient, and proved to be suitable for bioequivalence evaluations of different formulations containing valsartan and mirtazapine, respectively.
引文
1．戴德哉主编．临床药理学．北京：中国医药科技出版社，2000，28．
2. Zhou SD, Song Q, Tang Y, et al. Critical review of development, validation, and transfer for high throughput bio analytical LC-MS/MS method. Current Pharmaceutical Analysis. 2005, 1: 3-14.
3．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．
4．孙毓庆主编．现代色谱法及其在医药中的应用．北京：人民卫生出版社，2005，196-203．
5．姚彤炜主编．体内药物分析．浙江：浙江大学出版社，2000，68．
6. Hout MW, Zeeuw RA, Franke JP, et al. Evaluation of programmed temperature vaporizer for large-volume injection of biological samples in gas chromatography. J Chromatogr B. 1999, 729: 199-210.
7．李发美主编．医药高效液相色谱技术．北京：人民卫生出版社，1999，3-8，23-37，19，92-96．
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. Lee MS, Kems EH. LC/MS applications in drug development. Mass Spectrom Rev. 1999, 18: 187-279.
10．安登魁主编．现代药物分析选论．北京：中国医药科技出版社，2000，446-450．
11．刘志广，张华，李亚明主编．仪器分析．大连：大连理工大学出版社，2004，356
12．余自成．生物等效性研究中的若干问题．中国临床药理学杂志．2002，19：62-66．
13．国家药典委员会编．药物制剂人体生物利用度和生物等效性试验指导原则．中华人民共和国药典，2005年版二部，北京：化学工业出版社．2005：附录173-176．
14．胡钢英．血管紧张素Ⅱ受体阻滞剂的临床研究现状．国外医学内科学分册，2001，28：43-62．
15. Macek J, Klima J, Ptacek P. Rapid determination of valsartan in human plasma by protein precipitation and high-performance liquid chromatography. J Chromatogr B. 2006, 832: 169-172.
16．世界药物新闻(六十)．天津药学．2002，14：77．
17. Prasad PP, Yeh CM, Gurrieri P, et al. Pharmacokinetics of multiple doses of valsartan in patients with heart failure. J Cardiovasc Pharmacol. 2002, 40: 801-807.
18．王伟．缬沙坦的药理学特点与临床应用．医药导报．2002，21：520-521．
19．喻东山．抗抑郁药与快速起效．河北精神卫生．2002，15：47-49．
20．李春元，李梓．抗抑郁新药的临床研究进展．国外医药．2001，22：143-146．
21．翁史曼，李华芬，赵靖平，等．米氮平与氟西汀治疗抑郁症的多中心对照研究．中国新药与临床杂志，2001，20：329-333．
22．方世平，杨宝玉．抗抑郁症新药米氮平的药理与临床．中国新药杂志．1999，8：165-167．
23. Manddoli R, Pucci V, Sabbioni C, et al. Enantioselective determination of the novel antidepressant mirtazapine and its active demethylated metabolite in human plasma by means of capillary electrophoresis. J Chromatogr A. 2004, 1051: 253-260.
24．刘红梅，刘艳，金锐，等．高效液相色谱法测定人血浆中缬沙坦的药物浓废。中国药房．2001，12：414-415．
25. Gonzalez L, Lopez JA, Alonso RM, et al. Fast screening method for the determination of angiotensin Ⅱ receptor antagonists in human plasma by high-performance liquid chromatography with fluofimetric detection, J. Chromatogr A. 2002, 949: 49-60.
26. Sechaud R, Graf P, Bigler H, et al. Bioequivalence study of a valsartan tablet and a capsule formulation after single dosing in healthy volunteers using a replicated crossover design, Int J Clin Pharmacol Ther. 2002, 40: 35-40.
27. Nie J, Zhang M, Fan Y, et al. Biocompatible in-tube solid-phase microextraction coupled to HPLC for the determination of angiotensin Ⅱ receptor antagonists in human plasma and urine, J Chromatogr B. 2005, 828: 62-69.
28．许长江，刘骁，王海学，等．缬沙坦胶囊在人体的药物动力学及相对生物利用度．中国临床药学杂志．2002，11：91-94．
29．徐燕丰，曹文，林欣，等．高效液相色谱法测定血浆缬沙坦浓度．中国药理学通报．2002，18： 586-588.
30. Daneshtalab N, Lewanczuk RZ, Jamali F. High-performance liquid chromatographic analysis of angiotensin Ⅱ receptor antagonist valsaxtan using a liquid extraction method. J Chromatogr B. 2002, 766: 345-349.
31．赵志刚，李晔，陈旭，等．缬沙坦胶囊生物等效性研究．中国药学杂志．2002，37：686-689．
32．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．
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. Romoguieres T, Pehourcq F, Matoga M, et al. Determination of mirtazapine and its demethyl metabolite in plasma by high-performance liquid chromatography with ultraviolet detection. Application to management of acute intoxication. J Chromatogr B. 2002, 775: 163-168.
35. Dodd S, Burrows GD, Norman TR. Chiral determination of mirtazapine in human blood plasma by high-performance liquid chromatography. J Chromatogr B. 2000, 748: 439-443.
36. Meine L, Kress L, Posser W, et al. Therapeutic drug monitoring of mirtazapine and its metabolite desmethylmirtazapine by HPLC with fluorescence detection. Ther Drug Monit. 2004, 26: 277-283.
37. Ptacek P, Klima J, Macek J. Determination of mirtazapine in human plasma by liquid chromatography. J Chromatogr B. 2003, 794: 323-328.
38. Maris FA, Dingler E, Niehues S. High-performance liquid chromatographic assay with fluorescence detection for the routine monitoring of the antidepressant mirtazapine and its demethyl metabolite in human plasma. J Chromatogr B. 1999, 721: 309-316.
39. Labat L, Deveaux M, Dallet P, et al. Separation of new antidepressants and their metabolites by micellar electrokinetic capillary chromatography. J Chromatogr B. 2002, 773: 17-23.
40. Paus E, Jonzier PM, Cochard N, et al. Chirality in the new generation of antidepressants: stereoselective analysis of the enantiomers of mirtazapine, N-demethylmirtazapine, and 8-hydroxymirtazapine by LC-MS. Ther Drug Monit. 2004, 26: 366-374.
