氟马西尼有关物质的研究与测定

Research and determination of related substances in flumazenil

建立高效液相色谱法采用校正因子测定氟马西尼原料中有关杂质。采用Agilent Pursuit XRs C18(250 mm×4.6 mm,5μm)色谱柱,以稀磷酸溶液、甲醇和四氢呋喃为流动相,等度洗脱。采用标准曲线法测定校正因子,通过校正因子计算杂质含量。并用外标法验证校正因子方法的合理性、准确性和可行性。通过强制降解制备含有降解产物的氟马西尼降解溶液;采用LC-MS对降解产生的杂质进行结构确证。结果表明,氟马西尼和各杂质之间均分离良好,杂质A、杂质B、杂质D和杂质E的定量限分别为0.1699、0.3147、0.1439和0.2708 ng,检测限分别为0.0558、0.0969、0.0488和0.0890 ng,质量浓度分别在0.0349~7.8470、0.0387~8.7107、0.0346~7.7941和0.0324~7.2928μg·mL^(-1)内,线性关系良好(n=7)。平均回收率在98.25%~99.42%之间,相对标准偏差(RSD)均小于2.0%(n=9),方法的准确度高。采用外标法和校正因子法测定氟马西尼有关物质,两种方法结果一致。建立的HPLC方法准确度和灵敏度高、重复性好,可用于氟马西尼有关物质的测定。

A high performance liquid chromatography(HPLC)method utilizing correction factors was established for the quantitative detection of related substances in flumazenil.Separation was achieved using an Agilent Pursuit XRs C18 column(250 mm×4.6 mm,5μm)with an isocratic elution of dilute phosphoric acid,methanol,and tetrahydrofuran as the mobile phases.Correction factors calculated from a standard curve method were applied to determine the impurity content.The quantification of impurities in flumazenil was conducted using both external standard and correction factor methods,followed by validation and comparison of the two.For the identification of degradation products,a forced degradation approach was employed to prepare a flumazenil degradation solution,and the resulting impurities were confirmed by LC-MS analysis.The separation of flumazenil and its impurities was found to be efficient.The limits of quantification for impurities A,B,D,and E were established at 0.1699,0.3147,0.1439,and 0.2708 ng,respectively,with the limits of detection at 0.0558,0.0969,0.0488,and 0.0890 ng.These impurities demonstrated a strong linear relationship across the concentration ranges of 0.0349−7.8470,0.0387−8.7107,0.0346−7.7941,and 0.0324−7.2928μg·mL^(-1),respectively(n=7).The method achieved average recoveries between 98.25%and 99.42%,with an RSD of less than 2.0%(n=9),indicating high accuracy.The external standard and correction factor methods were used to determine the related substances in flumazenil,and the results of the two methods were consistent.The established HPLC method is characterized by its high accuracy,sensitivity,and repeatability,and is suitable for determining related substances in flumazenil.