LC-MS/MS法测定替诺福韦前体药物中遗传毒性杂质

Determination of genotoxic impurities impurities in Tenofovir disoproxil prodrug by LC-MS/MS

目的采用高效液相色谱-三重四极杆质谱法(LC-MS/MS)法测定替诺福韦前体药物中遗传毒性杂质9-丙烯基腺嘌呤含量,并分析最小二乘法不同线性拟合方法对结果准确度的影响。方法采用Waters XBridge C_(18)(4.6 mm×150 mm,3.5μm)色谱柱;以甲醇-水(55∶45)为流动相,流速:1.0 mL·min^(-1),等度洗脱8 min(2.0~2.6 min进质谱)。进样体积2μL,柱温40℃。采用正离子电喷雾(ESI^(+))模式电离,多反应离子监测(MRM)模式,选择m/z 176.0→136.0作为检测离子。结果经分析方法验证,该方法专属性良好;系统精密度试验RSD为1.1%(n=6);使用最小二乘法进行线性回归,线性范围0.051~25.250μg·mL^(-1);加权线性回归在低浓度区域准确性明显高于未加权线性回归。定量限0.051 ng·mL^(-1),检出限0.017 ng·mL^(-1);原料药样品溶液平均回收率99.08%~99.97%(n=9),重复性RSD为0.4%~1.3%;片剂样品溶液平均回收率98.94%~101.96%(n=9),重复性RSD为0.2%~0.3%;耐用性良好。结论建立的方法操作简单、灵敏度高、分析速度快、基质不影响检测,结果准确可靠,能够满足痕量遗传毒性杂质的检测要求。

Objective To determine the content of genotoxic impurity 9-propenyladenine in the prodrug of Tenofovir disoproxil by high performance liquid chromatography triple quadrupole mass spectrometry(LC-MS/MS),and analyze the impact of different curve fitting methods on the accuracy of the results.Methods Waters XBridge C 18 column(4.6 mm×150 mm,3.5μm)was used with methanol-water(55∶45)as the mobile phase,the flow rate was 1.0 mL·min^(-1).The injection volume was 2μL,and the column temperature was 40℃.Electrospray ionization source was used positive ion mode and multiple reaction monitoring(MRM)mode was performed with transitions of m/z 176.0→136.0.Results The results had been validated by analytical methods,and the specificity of this method was good;System precision test RSD=1.1%(n=6);The least square method was used for linear regression,the linear range was 0.051~25.250μg·mL^(-1);The accuracy of weighted linear regression in low concentration areas was significantly higher than that of unweighted linear regression.The quantitative limit concentration was 0.051 ng·mL^(-1),the detection limit concentration was 0.017 ng·mL^(-1);The average recovery rate of the raw material drug sample solution was 99.08%~99.97%(n=9),and the repeatability RSD was 0.4%~1.3%;The average recovery rate of the tablet sample solution was 98.94%~101.96%(n=9),and the repeatability RSD was 0.2%~0.3%;The tolerance was good.Conclusion The established method was characterized by simple operation,high sensitivity,fast analysis speed,and the matrix did not affect the detection.The results were accurate and reliable,and can meet the detection requirements for trace genotoxic impurities.