反相高效液相色谱法测定重组人溶菌酶的含量及纯度

目的：建立重组人溶菌酶含量及纯度的反相高效液相色谱（RP-HPLC）检测方法。方法：采用Welch Ultimate XB-C₁₈色谱柱（4.6 mm×250 mm，5 μm），含0.1%三氟乙酸和0.9%氯化钠的水溶液为流动相A；含0.1%三氟乙酸和0.9%氯化钠水溶液-乙腈（40：60）为流动相B；检测波长280 nm，柱温32℃，流速1.0 mL·min^-1，线性梯度洗脱。结果：本方法专属性良好；供试品48 h内稳定，RSD为0.51%；重复性RSD为0.46%，中间精密度不同工作日间的RSD为0.52%，不同实验员间的RSD为0.53%；定量下限200 ng、检测下限30 ng；在进样量10~60 μg范围内，线性关系良好，r=0.999 9；平均回收率（n=9）99.7%，RSD为0.94%。测定3批重组人溶菌酶冻干粉含量分别为36.5%、32.8%和31.1%；纯度均大于98%。结论：方法学验证表明，该方法符合定量检测重组人溶菌酶含量和纯度的要求，适用于重组人溶菌酶的质量控制。

Objective: To establish a method to determine (recombinant human lysozyme rhLYZ) by reversed phase high performance liquid chromatography(RP-HPLC),including quantitative analysis and purity analysis. Methods: Chromatography was performed on Welch Ultimate XB-C₁₈ chromatographic column(4.6 mm×250 mm, 5μm),using 0.1% trifluoroacetic acid and 0.9% sodium chloride solution as mobile phase A;and 0.1% trifluoroacetic acid and 0.9% sodium chloride solution-acetonitrile(40:60),as mobile phase B,with linear gradient elution at a flow rate of 1.0 mL·min^-1. The detection wavelength was 280 nm,and the column temperature was 32℃. Results: The method has a good specificity. The sample solution is stable within 48,RSD was 0.51%. Repetitive RSD was 0.46%,the intermediate precision(RSD) at different workdays was 0.52% and the RSD was 0.53% from different lab technicians. Quantitative limit was 200 ng and detection limit was 30 ng. In a sample range of 1060 μg,linearity was good,r was 0.999 9. The average recovery was 99.7%(n=9),with RSD of 0.94%. The content of three batches of rhLYZ were 36.5%,32.8% and 31.1%,respectively,with purity greater than 98%. Conclusion: Methodology validation shows that this method conforms to requirement of the quantitative determination of rhLYZ content and purity,and an be applied to quality control.