青霉素杂质谱分析方法的优化与转换

目的建立青霉素杂质谱HPLC分析方法,并将其转换成UPLC/UHPLC方法。方法以青霉素混合降解溶液为样品;首先分析《中国药典》(2010年版,ChP2010)方法甲醇-缓冲盐二元流动相色谱系统的缺陷;再利用实验设计理念,以响应曲面法(response surface methodology,RSM)的中心组合设计(central composition design,CCD)对色谱系统进行优化,满意度函数法确定最优色谱条件,并优化梯度洗脱条件;最后,利用软件对HPLC方法的流速、进样体积和梯度时间进行几何缩放,并通过色谱柱的选择,将其分别转换为UPLC方法和UHPLC方法。结果新HPLC方法:色谱柱为Capcell Pak C18 MGII(4.6mm×250mm,5μm),流速:1.0m L/min,进样体积:20μL;UPLC方法:色谱柱为Cortecs C18(2.1mm×100mm,1.6μm),流速:0.35m L/min,进样体积:2.0μL;UHPLC方法:Cortecs C18(4.6mm×150mm,2.76μm),流速:0.8mL/min,进样体积:10μL。3种方法的检测波长均为225nm,柱温均为34℃;流动相A均为磷酸盐缓冲液(取磷酸二氢钾10.6g,加水至1000mL,用磷酸调pH至3.4)-甲醇(72:14,V/V),流动相B均为乙腈;均为梯度洗脱,但梯度洗脱表不同。结论 3个色谱系统的分离效果(出峰顺序和个数)相似。新HPLC方法可以分离出更多的降解杂质,并明显改善了青霉素峰的拖尾,缩短了分析时间。

The optimization and transformation of analytical methods for the impurity profiling of penicillin

Objective To develop an HPLC method to analyze the related substances in penicillin and transfer the HPLC method into an UPLC/UHPLC method. Methods The mixed degradation solution of penicillin was used as the analytical sample. Firstly, the shortages of the analytical method in Chinese Pharmacopoeia(ChP2010) were shown, which applies methanol and phosphate buffer as mobile phase. This chromatographic system was optimized using the central composition design of response surface methodology with the concept of design of experiment(DoE). The best chromatographic condition was determined using the desirability function method. The gradient elution procedure was also optimized. Finally, the flow rate, the injection volume, and the gradient time of HPLC were geometrically scaled by software, and the HPLC method was transformed into UPLC and UHPLC methods through the selection of chromatographic column. Results The condition of the new developed HPLC system was as follows: the column used was Capcell Pak C18 MGII(4.6 mm×250 mm, 5μm), the flow rate was 1.0 mL/min and the injection volume was 20μL;the condition of the new developed UPLC system was as follows: the column used was Cortecs C18(2.1 mm×100 mm, 1.6μm), the flow rate was 0.35 mL/min and the injection volume was 2.0μL;the condition of the new developed UHPLC system was as follows: the column used was Cortecs C18(4.6 mm×150 mm, 2.76μm), the flow rate was 0.8 mL/min and the injection volume was 10μL. Other conditions of the three methods were all the same. The detection wavelength was 225 nm, the column temperature was 34℃, the mobile phase A was phosphate buffer(take 10.6 g of potassium dihydrogen phosphate, add water to 1000 mL, and adjust the pH to 3.4 with phosphoric acid)-methanol(72:14, V/V) and the mobile phase B was acetonitrile. But the three gradient elution procedures were different. Conclusion The separation performance(elution order and the number of peaks) of the three methods was similar. The new HPLC method can separate more degradation impurities, improve the trailing of penicillin and decrease the analytical time.