目的 建立纤溶酶反相高效液相色谱纯度测定方法。方法 采用SHISEIDO CAPCELL PAK C₁₈ SG300（250 mm×4.6 mm，5 μm），以0.1%三氟乙酸溶液-乙腈（95：5）为流动相A，以0.08%三氟乙酸溶液-乙腈（20：80）为流动相B，进行梯度洗脱，体积流量为0.7 mL·min^-1，检测波长280 nm，柱温40℃。参照2020年版《中国药典》四部9101药品质量标准分析方法验证指导原则进行专属性、检测限、耐用性考察。应用所建立的方法和分子排阻色谱法对3批样品纯度进行检测。结果 建立的反相色谱法专属性强，检测限为0.01%，耐用性强。对3批样品进行检测，质量分数在93.5%~96.9%，结果准确。结论 建立反相色谱法测定纤溶酶纯度，分辨率更高、重现性更好，可进行更好的质量控制。

Objective To establish a method of reversed-phase high performance liquid chromatography to determine the purity of fibrinolytic enzyme. Methods Using SHISEIDO CAPCELL PAK C₁₈ SG300 (250 mm×4.6 mm, 5 μm) or equivalent performance column. The mobile phase A was 0.1% trifluoroacetic acid solution - acetonitrile (95: 5), and the mobile phase B was 0.08% trifluoroacetic acid solution: acetonitrile (20:80) with gradient elution at the flow rate of 0.7 mL·min^-1, the detection wavelength was 280 nm, and the column temperature was 40 ℃. The specificity, detection limit and durability were investigated by referring to the guiding principles of drug quality standard analysis method validation of IV 9101 of Chinese Pharmacopoeia (2020 edition). The method and molecular exclusion chromatography were used to detect the purity of three batches of samples. Results The established reversed-phase chromatography has strong specificity, detection limit of 0.01% and durability. Three batches of samples were tested, the mass fraction was 93.5%—96.9%, and the results were accurate. Conclusion A reversed-phase chromatography method was established for the determination of plasminase purity with higher resolution, better reproducibility and better quality control.

R917