HPLC-CAD法测定乳糖含量及有关物质

目的：针对目前普遍反映的国内外药典中乳糖的含量和有关物质测定方法灵敏度低、硅胶基质的氨基色谱柱因流失严重而耐用性较差的问题，尝试采用聚合物基质的氨基柱，建立适合乳糖含量及有关物质测定的高效液相色谱-电喷雾检测器（HPLC-CAD）方法。方法：采用Shodex Asahipak NH₂P-50 4E色谱柱（4.6 mm×250 mm，5 μm），柱温30℃，流动相为乙腈-水（75∶25），流速1.0 mL·min^-1，供含量及有关物质测定用溶液的质量浓度分别为20 μg·mL^-1和2.0 mg·mL^-1，电喷雾检测器参数为：雾化温度35℃，采样频率10 Hz，幂率（PFV）参数1.15。结果：乳糖与相邻杂质以及各杂质峰间的分离均良好；乳糖质量浓度在1.0~100.4 μg·mL^-1范围内线性关系良好（r=0.999 8）；方法检测限（S/N=3）为8.03 ng，定量限（S/N=10）为20.08 ng；进样精密度RSD为1.1%；重复性RSD为0.90%；色谱柱在连续进样超过300次后，柱效没有明显降低。结论：所建方法灵敏度明显优于现行方法，选用的色谱柱耐用性明显提高，弥补了现有国内外药典中乳糖的含量和有关物质测定方法的不足，可作为乳糖质量控制新方法。

Objective:In order to solve the problem of the poor sensitivity and durability of present methods for the detection of lactose and its related substances with the silicone amino column in domestic and international pharmacopoeia,we established a suitable HPLC-CAD method using a polymer matrix amino column for the determination of lactose and its related substances.Methods:A Shodex Asahipak NH₂P-50 4E (4.6 mm×250 mm,5 μm)column was adopted.The column temperature was set at 30℃.The mobile phase consisted of acetonitrile and water (75:25)at a flow rate of 1.0 mL·min^-1.Concentrations of solution for the determination of content and related substances were 20 μg·mL^-1 and 2.0 mg·mL^-1,respectively.The detector of CAD was applied with nebulization temperature at 35℃,and collection frequency at 10 Hz,and power function value (PFV)at 1.15. Results:Lactose and all impurities were well separated by this method.The linearity of lactose was fine in the range of 1.0-100.4 μg·mL^-1 with the correlation coefficient of 0.999 8.The LOD (as S/N=3)was 8.03 ng and the LOQ (as S/N=10)was 20.08 ng.The RSDs of precision and repeatability were 1.1% and 0.90%,respectively.The column efficiency of this column was not significantly reduced after more than 300 injections in a row. Conclusion:The new established HPLC-CAD method shows better sensitivity and robustness of determination than that of existing methods and can make up for the defects of present domestic and international pharmacopoeia methods,which could be a reliable method for the quality control of lactose.