目的 建立反相高效液相色谱法(RP-HPLC)测定溶菌酶的纯度。方法 使用Agilent ZORBAX 300SB C₈色谱柱(4.6 mm×250 mm,5 μm);以三氟乙酸-水-乙腈(2∶700∶300)为流动相A,以三氟乙酸-水-乙腈(2∶200∶800)为流动相B,进行梯度洗脱,流速为1.0 mL·min^-1,检测波长为280 nm,进样量为20 μL。并采用QE Plus质谱仪对其中单个最大杂质进行了相对分子质量测定。结果 建立的PR HPLC方法能够有效分离溶菌酶中主要杂质,主峰与各杂质峰均能较好分离,方法专属性强、溶菌酶在0.004～0.5 mg·mL^-1的内线性良好(r=1.000 0);定量限为0.08 μg,检出限为0.04 μg;精密度、重复性、20 h样品稳定性实验的相对标准偏差(RSD)值均小于0.5%;11批样品纯度测定结果为89%～94%;各样品中单个最大杂质峰中所含物质是相对分子质量不等的混合物。结论 建立的RP-HPLC方法可以用于溶菌酶纯度的检测。

OBJECTIVE To establish a RP HPLC method of purity for lysozyme. METHODS The chromatographic separation was performed on a Agilent ZORBAX 300SB C₈ column(4.6 mm×250 mm, 5 μm)with trifluoroacetic acid-water-acetonitrile (2∶700∶300) as mobile phase A, and trifluoroacetic acid-water-acetonitrile (2∶200∶800) as mobile phase B in a gradient elution. The flow rate was 1.0 mL·min^-1 and the UV detection wavelength was set at 280 nm.the injection volume was 20 μL. The molecular weight of the maximum impurity was detected using QE Plus. RESULTS Under the selected chromatographic conditions, many impurities can be separated, the main peak and each impurity peak can be separated well. The linear range of lysozyme was 0.004-0.5 mg·mL^-1(r=1.000 0). The limit of quantification was 0.8 μg and the limit of detection was 0.4 μg.The RSDs of precision, repeatability and stability in 20 hours were all less than 0.5%. The purity range of 11 samples was 89%-94%. The substances contained in the maximum impurity are mixtures of different molecular weights. CONCLUSION The method is suitable for the detection of lysozyme purity.