Quantitative analysis of sucrose in human fibrinogen by HPLC / 中国药学杂志

ABSTRACT

OBJECTIVE: To establish a quantitative determination method of sucrose in human fibrinogen by HPLC. METHODS: An HPLC method was developed to specifically determine sucrose on Zorbax carbohydrate analysis column with Waters Alliance system and 2414refractive index detector. The separation was performed at 30°C using a mobile phase consisting of acetonitrile and water (7030) at a flow rate of 1.4 mL · min-1. Thirteen batches of recombinant human coagulation factor VM samples were selected for methodology comparison of the established HPLC method with the IEC-HPLC method adopted by Ch. P 2010, because these samples only contained sucrose and did not receive dry-heat treatment. RESULTS: The RSDs (n=6) of the retention time and peak area were 0.17% and 0.09%, respectively. The recoveries of sucrose at low (5 mg · mL-1), middle (10 mg · mL-1), high (15 mg · mL-1) concentration were 96.2%, 98.8% and 100.3%, respectively. The average recovery was 98.4% and the linear correlation coefficient r was 1.0000 in the ranges of 2-20 mg · mL-1. Statistical analysis showed that the amounts of sucrose in 13 batches of recombinant human coagulation factor VIII samples determined by the HPLC method and IEC-HPLC method had no significant difference (P≤0.05) and the correlation coefficient r was 1.0000. CONCLUSION: The proposed HPLC method is simple, accurate and re-peatable for determination of sucrose in dry-heat treated human fibrinogen product. The HPLC method showed good accordance with IEC-HPLC method for assay results of sucrose, thus can be widely applied.