高效液相色谱法定量测定人纤维蛋白原中蔗糖含量

Quantitative Analysis of Sucrose in Human Fibrinogen by HPLC

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。

OBJECTIVE To establish a quantitative determination method of sucrose in human fibrinogen by HPLC. METH- ODS 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 co- efficient 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 re- combinant 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 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.