高效液相色谱法定量测定人纤维蛋白原中蔗糖含量

 目的 建立定量测定人纤维蛋白原中蔗糖含量的HPLC分析方法，对2010年版《中国药典》相关测定方法进行有益补充。方法 采用Waters Alliance系统、Waters 2414示差折光检测器、安捷伦Zorbax Carbohydrate Analysis色谱柱，以ACN-H₂O（70∶30）作为流动相，流速1.4 mL·min^-1，柱温30 ℃测定人纤维蛋白原中的蔗糖。并用该方法平行测定13批含有蔗糖的重组人凝血因子Ⅷ，与药典IEC-HPLC方法进行方法学比较。结果 HPLC测定蔗糖对照品的保留时间RSD (n=6)为0.17%，峰面积RSD (n=6 )为0.09%。蔗糖在低（5 mg·mL^-1)、中（10 mg·mL^-1)、高（15 mg·mL^-1)3个质量浓度的回收率分别为96.2% ，98.8% ，100.3%，平均回收率为98.4%。蔗糖在2~20 mg·mL^-1（r=1.000 0）内与峰面积呈良好的线性关系。2批人纤维蛋白原中的蔗糖含量分别为48.8，45.0 g·L^-1，均符合其40~60 g·L^-1的质量范围。平行测定的13批重组人凝血因子Ⅷ的蔗糖结果与药典IEC-HPLC测定结果的相关系数r为1.000 0，两种方法无显著性差异（P>0.05）。结论 新建HPLC测定蔗糖含量方法的准确性和精密度良好，能够规避药典IEC-HPLC色谱条件下枸橼酸和蔗糖互相干扰的问题，本法可以适用于干热病毒灭活条件下人纤维蛋白原中蔗糖检测。

Quantitative Analysis of Sucrose in Human Fibrinogen by HPLC

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 2414 refractive index detector. The separation was performed at 30 ℃ using a mobile phase consisting of acetonitrile and water (70∶30) at a flow rate of 1.4 mL·min^-1. Thirteen batches of recombinant human coagulation factor Ⅷ 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 1.000 0 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 1.000 0. CONCLUSION The proposed HPLC method is simple，accurate and repeatable 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.