高效液相色谱加校正因子主成分对照法测定阿哌沙班片中有关物质的含量

Determination of related substances in apixaban tablets by HPLC-principal component self-control with correction factor

目的建立高效液相色谱加校正因子的主成分自身对照法同时测定阿哌沙班片中12个有关物质(杂质A~L)的含量并对其限度进行探讨。方法采用Waters Xbridge C_(18)(250 mm×4.6 mm,5μm)色谱柱,以30 mmol·mL^(-1)醋酸铵缓冲液(pH 4.50)为流动相A,乙腈为流动相B,梯度洗脱,流速1.0 mL·min^(-1),柱温40℃,检测波长280 nm,进样体积10μL。绘制阿哌沙班和12个杂质的标准曲线,以斜率计算杂质相对于阿哌沙班的校正因子,并采用Nexus 2.6.0软件对各杂质进行遗传毒性预测。结果阿哌沙班与各杂质的色谱峰均能良好分离,且在相应范围内与峰面积均呈良好线性关系(r>0.999)。阿哌沙班与12个杂质的定量限在0.0396~0.0582μg·mL^(-1)。杂质A~L低、中、高浓度水平的平均回收率(n=3)分别为101.77%~102.60%、101.82%~103.32%、100.10%~102.09%、99.55%~101.31%、99.91%~103.12%、100.36%~102.93%、100.86%~102.03%、101.25%~102.44%、103.13%~104.58%、100.33%~100.84%、100.17%~102.65%和100.46%~103.03%。采用加校正因子的主成分自身对照法和外标法测定杂质含量的结果差异无统计学意义(P>0.05)。遗传毒性软件预测结果所有杂质均为阴性(ICH M7第5类)。结论本方法灵敏度高、专属性强、准确性好,可用于阿哌沙班片的有关物质检查。

Objective To establish an HPLC-principal component self-control with correction factor method to simultaneous determine 12 related substances(impurities A~L)in apixaban tablets and their limit values.Methods The determination was performed on a Waters Xbridge C_(18)column(250 mm×4.6 mm,5μm).The mobile phase consisted of 30 mmol·mL^(-1)ammonium acetate buffer solution(pH 4.50)(phase A)and acetonitrile(phase B)with gradient elution at a flow rate of 1.0 mL·min^(-1).The column temperature maintained at 40℃,and the detection wavelength was set at 280 nm.The injection volume was 10μL.The linear curve of apixaban and the 12 impurities were drawn and the correction factors of each impurity related to apixaban were calculated by slope.The genotoxicity of the impurities was predicted by Nexus 2.6.0 software.Results Apixaban and the 12 impurities were well separated.The linearity of apixaban and 12 impurities was good within the corresponding ranges(r>0.999).The limit of quantification was 0.0396~0.0582μg·mL^(-1).The average recoveries of low,medium and high concentrations(n=3)of impurities A~L were 101.77%~102.60%,101.82%~103.32%,100.10%~102.09%,99.55%~101.31%,99.91%~103.12%,100.36%~102.93%,100.86%~102.03%,101.25%~102.44%,103.13%~104.58%,100.33%~100.84%,100.17%~102.65%and 100.46%~103.03%,respectively.The impurity content measured by principal component self-control with correction factor method and the external standard method showed no significant difference(P>0.05).The predicted genotoxicity of all the impurities by Nexus 2.6.0 software was negative(ICH M7 class 5).Conclusion This method is sensitive,specific and accurate,which can be used for the determination of related substances in apixaban tablets.