【作者】 付淑军；

【导师】 钟大放； 陈笑艳；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2007， 硕士

【摘要】 目的：建立专属、灵敏的LC／MS／MS法，测定人血浆中奥司他韦、伊曲康唑和二者活性代谢物并用于临床药动学研究和制剂生物等效性评价。方法：同时测定人血浆中奥司他韦及其活性代谢物（Ro 64-0802）时，200μL，血浆样品经沉淀蛋白处理后，以甲醇：2mmol／L醋酸铵：甲酸（60：40：0.1，v／v／v）为流动相，Zorbax XDBC₁₈柱分离，采用API4000液相色谱-串联质谱仪，电喷雾电离源，正离子方式进行多反应监测（MRM）。用于定量分析的离子反应分别为m／z313→m／z225（奥司他韦），m／z285→m／z197（Ro 64-0802）和m／z 256→m／z 167（内标，苯海拉明）。同时测定人血浆中伊曲康唑和羟基伊曲康唑时，100μL血浆样品经沉淀蛋白处理后，以乙腈：水：甲酸（80：20：0.2，v／v／v）为流动相，Zorbax SB C₁₈柱分离，采用Thermo FinniganQuantum Ultra液相色谱-串联质谱仪，电喷雾电离源，以选择反应监测（SRM）方式进行正离子检测。用于定量分析的离子反应分别为m／z705→m／z（392+432）（伊曲康唑），m／z721→m／z（392+408）（羟基伊曲康唑）和m／z 256→m／z 167（内标，苯海拉明）。结果：测定人血浆中奥司他韦和Ro 64-0802的线性范围分别为0.435-435 ng／mL和0.740—740ng／mL：二者日内、日间精密度（RSD）均小于11.9％，准确度（RE）均在±3.8％以内。该方法已成功应用于磷酸奥司他韦制剂的临床生物等效性评价。测定人血浆中伊曲康唑及羟基伊曲康唑的线性范围均为1.00-1000ng／mL，定量下限均为1.00ng／mL。日内、日间精密度（RSD）均小于14.4％，准确度（RE）均在±6.3％以内。该方法已成功应用于伊曲康唑及羟基伊曲康唑的药物动力学研究。结论：所建立的LC／MS／MS法专属、灵敏、简便、快速，可用于奥司他韦、伊曲康唑和二者活性代谢物的血药浓度测定并进行临床药动学研究及制剂的生物等效性评价。更多还原

【Abstract】 Objective: To develop and validate rapid, sensitive and specific methods for quantitative analyses of oseltamivir and its active metabolite Ro 64-0802, itraconazole （ITZ） and its active metabolite hydroxyitraconazole （HIT） in plasma by liquid chromatographic-tandem mass spectrometry for pharmacokinetic studies.Method: Oseltamivir, Ro 64-0802 and internal standard diphenhydramine were simply pretreated by protein precipitation using acetonitrile, and then analyzed on a Zorbax XDB C₁₈ column. The mobile phase consisted of methanol-2 mM ammonium acetate-formic acid（60: 40: 0.1, v/v/v）, at a flow-rate of 0.70 mL/min. An API 4000 tandem mass spectrometer equipped with turbospray ionization source was used as detector and was operated in the positive ion mode. Multiple reaction monitoring（MRM） using the precursor to product ion combinations of m/z 313→m/z 225, m/z 285→m/z 197 and m/z 265→m/z 167 was performed to quantify oseltamivir, Ro 64-0802 and the internal standard, respectively. ITZ, HIT and internal standard diphenhydramine were extracted from plasma using protein precipitation with acetonitrile, then separated on a Zorbax SB C₁₈ column. The mobile phase consisted of acetonitrile-water-formic acid （80: 20: 0.2, v/v/v）, at a flow-rate of 0.50 mL/min. A Thermo Finnigan TSQ Quantum Ultra tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring using（SRM） the precursor to product ion combinations ofm/z 705→m/z （392+432）, m/z 721→m/z （392 + 408） and m/z 256→m/z 167 was performed to quantify ITZ, HIT and the internal standard, respectively.Results: The linear of oseltamivir and Ro 64-0802 curves were obtained in the concentration ranges of 0.435 - 435 ng/mL and 0.740 - 740 ng/mL respectively. The inter- and intra- day precision （RSD） were below 11.9 %, and the accuracy （RE） were within±3.8% calculated from QC samples. The method was successfully used in bioequivalence study of oseltamivir and Ro 64-0802 in human plasma after oral administration of 75 mg oseltamivir. The linear concentration ranges of calibration curves for ITZ and HIT were both 1.00 - 1000 ng/mL. The inter- and intra- day precision （RSD） were below 14.4 %, and the accuracy （RE） were within±6.3 % calculated from QC samples. The method was successfully used in pharmacokinetic studies of ITZ and HIT in human plasma after oral administration of 200 mg itraconazole.Conclusion: The methods were successfully applied for the evaluation of the pharmacokinetics of oseltamivir, itraconazole and their metabolites.更多还原