Objective: To establish a determination method for molecular fraction ratio in the sample pretreatment of urokinase for injection using ion exchange chromatography(IEC) combined with size exclusion chromatography(SEC) so as to find a solution to fix the ratio of molecular fraction could not be controlled in the preparation's quality standard for a long time, the product quality of six domestic companies was investigated. Methods: Human serum albumin was removed by IEC for its distinguishing isoelectric point, and then the urokinase in the rest sample were measured by SEC. The chromatographic column was TSKgel G2000SWXL column(300 mm×7.8 mm, 5 μm) with 0.1 mol·L-1 sodium dihydrogen phosphate buffer(pH 3.0) as mobile phase, the flow rate was 0.5 mL·min-1, the column temperature was 35 ℃. The detection wavelength was 280 nm and the injection volume was 50 μL. Results: The urokinase at high and low molecular mass showed good linear relationship (r≥0.998 0) when the concentration of urokinase was in the range of 0.05 to 5.1 mg·mL-1. The detection limit of high molecular mass urokinase and low molecular mass urokinase were 1.5 μg·mL-1 and 5.1 μg·mL-1, respectively, and the limit of quantification were 5.1 μg·mL-1 and 16.9 μg·mL-1,respectively.The RSDs of precision, repeatability, stability test in 24 h were less than 4.0%. The recovery was 92.5%-97.0%, the relative content range of high molecular mass urokinase in all 18 batches of samples was 82.8%-96.5%, and low molecular mass urokinase was 3.5%-17.2%. Conclusion: The established ion exchange chromatography combined with size exclusion chromatography is applicable for the determination of molecular fraction in the preparation of urokinase for injection, and fills the gap in the current quality standards of urokinase for injection.
YAN Cui-xia, YIN Hong-rui, SHI Fang-liang, CHEN Gang, ZHENG Lu-xia, SHAO Hong
. Determination of molecular fraction ratio in urokinase for injection by ion exchange chromatography combined with size exclusion chromatography*[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(1)
: 4
-11
.
DOI: 10.16155/j.0254-1793.2023.01.01
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