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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-C_(18)色谱柱(4.6 mm×250 mm,5μm);流动相A为0.03mol·L~(-1)磷酸二氢钾(2 mol·L~(-1) Na OH调至p H7.5),流动相B为乙腈;柱温为35℃;检测波长为254 nm;流速为1.0 m L·min~(-1);液质联用方法采用XDB-C_(18)柱(4.6 mm×50 mm,1.8μm)色谱柱,以0.01 mol·L~(-1)甲酸铵缓冲溶液(p H6.5未调)-乙腈(90∶10)为流动相,检测波长为254 nm,流速为0.4 m L·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 k Pa。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·m L~(-1)范围内头孢匹胺钠呈良好的线性关系(R~2=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 a high performance liquid chromatography (HPLC) gradient elution method for the determination of cefpiramide-related substances, and to identify the structurally related substance I. METHODS: The mobile phase A was composed of ZORBAX SB-C 18 column (4.6 mm × 250 mm, 5 μm) and the mobile phase A was 0.03 mol·L -1 potassium dihydrogen phosphate (2 mol·L -1 Na OH was adjusted to p H7.5). The mobile phase B was acetonitrile, the column temperature was 35 ℃, the detection wavelength was 254 nm, the flow rate was 1.0 mL · min -1, and the liquid chromatography coupled with XDB-C_ 18 column (4.6 mm × 50 mm, 1.8 μm) column with 0.01 mol·L -1 ammonium formate buffer (p H6.5 not adjusted) -acetonitrile (90:10) as the mobile phase, The wavelength was 254 nm, the flow rate was 0.4 m L · min -1, and the column temperature was 30 ℃. The electrospray ionization (ESI) source was used with the detection mode of ESI (+), the drying gas flow rate was 10 L · min -1, the drying gas temperature was 340 ℃ and the atomizer was 275.8 kPa. Results: The peak of cefpiramide was separated well from its related substances. Cefpiramide sodium showed a good linear relationship (R ~ 2 = 1.000) in the range of 1.31 ~ 41.98 mg · m L -1. The quantitative method The limit of detection was 2.6 ng with a detection limit of 0.8 ng. LC-MS method was used to identify the structure of substance I as cefpiramide sodium isomers, and confirmed by the synthetic method of the structure is correct, the 7 side chain chiral atoms from R to S type. Conclusion: The method is rapid and accurate. The separation of starting materials, intermediates and main peaks, starting materials and intermediates is better than the standard methods and can be used for the determination of cefpiramide sodium, which is a powerful supplement to the current methods .