对比研究宽分布对照品测定依诺肝素钠分子量

Comparative Study on the Determination of Molecular Weight of Enoxaparin Sodium by Broad Distribution Reference Substance

摘要: 目的：对比研究欧洲药典(European Pharmacopeia, EP)和世界卫生组织(World Health Organization, WHO)低分子肝素分子量对照品(均为宽分布对照品，下称EP对照品和WHO对照品)测定依诺肝素钠分子量和分子量分布。方法：以TSK G2000SWxl (7.5 mm × 300 mm)为色谱柱，分别以0.2 mol•L−1硫酸钠水溶液(硫酸调pH值5.0)为流动相和0.1 mol•L−1醋酸铵水溶液为流动相，流速为0.5 mL•min−1，柱温30℃，紫外检测波长234 nm，示差折光检测器温度为35℃，进样量25 μL。结果：分别在两种流动相体系下，用EP对照品和WHO对照品，对依诺肝素钠CRS (EP，重均分子量标示量为4480 Da)、依诺肝素钠注射液(克赛®)、依诺肝素钠原料药(国产)和依诺肝素钠注射液(国产)，进行分子量和分子量分布检查，各样品重均分子量差值均小于100 Da，分子量分布差值均小于1%。结论：通过八批样品试验验证，EP对照品和WHO对照品用于依诺肝素钠分子量和分子量分布测定结果无显著性差异。

Abstract: Objective: Comparative study on the European Pharmacopeia (EP) and the World Health Organi-zation (WHO) low molecular weight heparin reference substances (RS) (both broad distribution reference substance, hereinafter referred to as EP RS and WHO RS), in the determination of the molecular weight and molecular weight distribution of enoxaparin sodium. Methods: A high per-formance size exclusion chromatography was applied by TSK G2000SWxl (7.5 mm × 300 mm) column. The mobile phase was 0.2 mol•L−1 sodium sulfate aqueous solution adjusted the pH value of 5.0 with sulfuric acid and 0.1 mol•L−1 ammonium acetate aqueous solution, respectively. An iso-cratic elution was performed at a flow rate of 0.5 mL•min−1, and the column temperature was maintained at 30˚C. UV detection with the wavelength at 234 nm was applied. A differential refrac-tion detector and its temperature was kept at 35˚C, with an injection of 25 μl was used. Results: By using EP RS and WHO RS in two different mobile phase systems, the molecular weight and molec-ular weight distribution of enoxaparin sodium CRS (weight-average molecular weight labeled as 4480 Da), enoxaparin sodium injection (clexane), enoxaparin sodium drug substance and enoxaparin sodium injection (self-produced) were determined. And according to the results, the difference of the weight-average molecular weight between samples was less than 100 Da, mean-while, the difference of the molecular weight distribution was less than 1%. Conclusion: The results of eight batches of samples showed that there was no significant difference in the determination of the molecular weight and molecular weight distribution for enoxaparin sodium via employing EP RS and WHO RS.