HPLC测定艾曲波帕中3,4-二甲基苯肼的残留量

Determination of residual 3,4-dimethylphenylhydrazine in Eltrombopag by HPLC

目的采用衍生化HPLC法测定艾曲波帕中的基因毒性杂质3,4-二甲基苯肼。方法用苯甲醛作为衍生化试剂,衍生化样品后,用HPLC法测定。采用Gemini C_(18)色谱柱(250 mm×4.6 mm,5μm),以0.1%三氟乙酸溶液-乙腈(30∶70)为流动相,流速1.0 mL·min^(-1),检测波长348 nm。结果3,4-二甲基苯肼衍生物与样品中其他组分能完全分离;0.02~0.12μg·mL^(-1)3,4-二甲基苯肼与峰面积的线性关系良好(r=0.9989),检测限为0.01μg·mL^(-1),进样精密度良好,3,4-二甲基苯肼衍生物在8 h内稳定(RSD=1.20%),平均回收率为102.10%,RSD为1.50%(n=9)。样品中未检出3,4-二甲基苯肼,其含量在1.0 ppm以下,符合要求。结论所用方法专属、灵敏、准确,适用于检测艾曲波帕中的基因毒性杂质3,4-二甲基苯肼。

OBJECTIVE To determine the residual 3,4-dimethylphenylhydrazine as a genotoxic impurity in Eltrombopag by HPLC.METHODS Benzaldehyde was used as the derivatization reagent.The separation was performed on a Gemini C_(18) column(250 mm×4.6 mm,5μm)with 0.1%trifluoroacetic acid solution-acetonitrile(30∶70)as mobile phase at a flow rate of 1.0 mL·min^(-1).The column temperature was 30℃,the detection wavelength was 348 nm,and the injection volume was 20μL.RESULTS The 3,4-dimethylphenylhydrazine derivatives was completely separated from other components in the sample.The 3,4-dimethylphenylhydrazine derivatives had good linearity in the range of 0.02-0.12μg·mL^(-1)(r=0.9989),and the limit of detection was 0.01μg·mL^(-1).The injection precision was validated;3,4-dimethylphenylhydrazine derivatives were stable within 8 h(RSD=1.2%).The average recovery was 102.1%with RSD of 1.5%(n=9).In the samples,3,4-dimethylphenylhydrazine was not detected,which meant the contents were below 1.0 ppm,meeting the requirements.CONCLUSION This method is specific,sensitive,accurate,and can be used for the detection of genotoxic impurity 3,4-dimethylphenylhydrazine in Eltrombopag.