HPLC梯度洗脱法测定头孢匹胺钠中有关物质

目的：建立用于检测头孢匹胺钠有关物质的高效液相色谱梯度洗脱法，并对其中关键的有关物质I进行结构鉴定。方法：采用ZORBAX SB-C₁₈色谱柱（4.6 mm×250 mm，5 μm）；流动相A为0.03 mol·L^-1磷酸二氢钾（2 mol·L^-1 NaOH调至pH7.5），流动相B为乙腈；柱温为35℃；检测波长为254 nm；流速为1.0 mL·min^-1；液质联用方法采用XDB-C₁₈柱（4.6 mm×50 mm，1.8 μm）色谱柱，以0.01 mol·L^-1甲酸铵缓冲溶液（pH6.5未调）-乙腈（90∶10）为流动相，检测波长为254 nm，流速为0.4 mL·min^-1，柱温为30℃。采用电喷雾ESI离子源，检测模式为ESI（+），干燥气流速为10 L·min^-1；干燥气温度为340℃，喷雾器为275.8 kPa。结果：头孢匹胺钠主峰与其相关物质之间分离良好，在1.31~41.98 mg·mL^-1范围内头孢匹胺钠呈良好的线性关系（R²=1.000），方法的定量限为2.6 ng，检测限为0.8 ng。采用LC-MS方法鉴定其中有关物质I的结构为头孢匹胺钠的异构体，并用合成方法确证了结构的正确性，其7位侧链手性原子由R型改变为S型。结论：本方法快速准确，起始原料、中间体与主峰，起始原料和中间体之间分离良好，优于现行标准方法，可用于头孢匹胺钠的有关物质检测，为现行方法的有力补充。

Objective: To establish an HPLC gradient elution method for the determination of the related substances of cefpiramide sodium, and to identify the key related substance I.Method: The HPLC method was carried out using a ZORBAX SB-C₁₈ column(4.6 mm×250 mm, 5 μm);Mobile phase A was 0.03 mol·L^-1 Potassiumdihydrogen phosphate solution(adjust pH to 7.5 with Sodium hydroxide), mobile phase B was acetonitrile;The column temperature was 35℃ and the detection wavelength was 254 nm;the flow rate was 1.0 mL·min^-1.The LC-MS method was carried out using an XDB-C₁₈ column(4.6 mm×50 mm, 1.8 μm);Mobile phase was 0.03 mol·L^-1 ammonium formate solution(pH6.5 not adjusted)-acetonitrile(90:10);The column temperature was 30℃ and the detection wavelength was 254 nm;the flow rate was 0.4 mL·min^-1.An electrospray ESI ion source was used to detect ESI(+), the drying airflow rate was 10 L·min^-1, the drying gas temperature was 340, and the sprayer was 275.8 kPa.Results: Between cefpiramide sodium peak and its related substances could be isolated.The good linear range of cefpiramide was 1.31-41.98 mg·mL^-1(R²=1.000), LOQ and LOD were 2.6 ng and 0.8 ng respectively.LC-MS was used to identify that the related substance I was isomer of cefpirmide sodium, and the structure was confirmed by synthetic method.The 7-side chain chiral atoms changed from R type to S type.Conclusion: The method is rapid and accurate.The separation of the starting materials, intermediates and main peaks, starting materials and intermediates is better than the existing standard method.It can be used for the detection of related substances of cefpiramide sodium, which is a powerful supplement to the present method.