柱前衍生-HPLC-FLD法测定对甲苯磺酸拉帕替尼中的基因毒性杂质

目的: 建立荧光胺柱前衍生-高效液相色谱-荧光检测法,测定对甲苯磺酸拉帕替尼中的基因毒性杂质3-氯-4-(3-氟苯基甲氧基)苯胺。方法: 3-氯-4-(3-氟苯基甲氧基)苯胺在pH 3.3条件下,与荧光胺反应生成荧光产物,采用高效液相色谱-荧光检测法测定,使用Kromasil l00-5C₁₈色谱柱(150 mm×4.6 mm,5 μm),以0.05 mol·L^-1醋酸铵缓冲液(pH 4.5)-乙腈(50:50)为流动相,流速1.3 mL·min^-1,荧光检测器激发波长290 nm,发射波长503 nm。结果: 线性范围为5~50 ng·mL^-1(r=0.996 1);定量限为2.0 ng·mL^-1(相当于0.000 04%);平均回收率(n=12)为96.4%;实验测得3批甲苯磺酸拉帕替尼中3-氯-4-(3-氟苯基甲氧基)苯胺含量分别为0.000 10%、0.000 08%与0.000 11%。结论: 建立的方法经方法验证,适用于对甲苯磺酸拉帕替尼中3-氯-4-(3-氟苯基甲氧基)苯胺的测定。

Objective: To develop a high performance liquid chromatographic method with fluorimetric detection after precolumn derivatization with fluorescamine for 3-chloro-4-(3-fluorobenzyloxy)-aniline(hereinafter called genotoxic impurity) determination in lapatinib ditosylate. Methods: The 3-chloro-4-(3-fluorobenzyloxy)-aniline in lapatinib ditosylate was derived with fluorescamine at pH 3.3,and analyzed by high performance liquid chromatography equipped with fluorescence detector.The Kromasil 100-5C₁₈ column(150 mm×4.6 mm,5 μm) was adopted for the analysis with a mobile phase consisting of 0.05 mol·L^-1 aqueous ammonium acetate (pH 4.5) and aceto nitrile (50:50) at the flow rate of 1.3 mL·min^-1,and the fluorescence detection was set λ_ex=290 nm and λ_em=503 nm. Results: The calibration curve was linear over the concentration range from 5 ng·mL^-1 to 50 ng·mL^-1 with the correlation coefficient of 0.996 1;the limit of quantity was 2.0 ng·mL^-1(equal to 0.000 04%),the average recovery(n=12) was 96.4%.The results of three batchs of samples were 0.000 10%,0.000 08% and 0.000 11%,respectively. Conclusion: The established method is proved to be suitable for the determination of 3-chloro-4-(3-fluorobenzyloxy)-aniline in lapatinib ditosylate.