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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 HPLC method for the quantitative determination of sucrose content in human fibrinogen and to supplement the relevant methods of determination of 2010 Chinese Pharmacopoeia. Methods Waters Alliance system, Waters 2414 refractive index detector and Agilent Zorbax Carbohydrate Analysis column were used. The mobile phase was ACN-H2O (70:30) with a flow rate of 1.4 mL · min-1 and the column temperature was set at 30 ℃ for determination of human fibrinogen In the sucrose. Thirteen batches of recombinant human coagulation factor VIII containing sucrose were determined by this method and compared with the method of Pharmacopoeia IEC-HPLC. Results The retention time RSD (n = 6) was 0.17% and the peak area RSD (n = 6) was 0.09%. The recoveries of sucrose were 96.2%, 98.8% and 100.3% at the concentrations of 5 mg.mL-1, 10 mg.mL-1 and 15 mg.mL-1, respectively The recovery rate was 98.4%. Sucrose showed a good linear relationship with the peak area in 2 ~ 20 mg.mL-1 (r = 1.000 0). The contents of sucrose in two batches of human fibrinogen were 48.8 and 45.0 g.L-1, respectively, which were within the mass range of 40-60 g.L-1. The correlation coefficients (r) between sucrose of 13 batches of recombinant human factor Ⅷ and the results of pharmacopoeia IEC-HPLC were 1.000 0. There was no significant difference between the two methods (P> 0.05). Conclusion The accuracy and precision of the new method for the determination of sucrose content by HPLC can avoid the interference of citric acid and sucrose under the conditions of the pharmacopoeial IEC-HPLC chromatogram. This method can be applied to human fibrinogen In sucrose testing.