阴离子交换HPLC法精确测定肝素钠中的多硫酸软骨素和硫酸皮肤素

Simultaneous determination of over-sulfated chondroitin sulfate and dermatan sulfate in heparin sodium by anion exchange HPLC

目的:建立可同时测定肝素钠中的多硫酸软骨素(OSCS)和硫酸皮肤素(DS)等杂质的有关物质检测方法。方法:应用高效液相色谱-紫外可见光检测器,2种阴离子交换树脂为填充剂的色谱柱,在pH 3.0的酸性体系下,设计了5种由不同流动相体系、洗脱梯度和不同阴离子交换柱组成的分析方法,比较了其分离度。结果:5种方法中以方法 E灵敏度最高,理论塔板数和分离度也最高,多硫酸软骨素的检测限和定量限分别为0.025%和0.03%;硫酸皮肤素的检测限和定量限分别为0.03%和0.05%;多硫酸软骨素和硫酸皮肤素的加标回收率分别为102.5%和96.0%。结论:采用方法 E的弱阴离子交换HPLC法与现有药典标准方法相比,具有更好的灵敏度和准确度。

Objective： To establish a precise method to simultaneously determine the related substances of over-sulfated chondroitin sulfate（OSCS）and dermatan sulfate（DS）in heparin products（HP）.Methods： VWD was applied,and the separation was performed on two kinds of anion exchanger columns：Dionex IonPac AS11（250 mm×2 mm,13 μm）and TSK-GEL DEAE-5PW（75 mm×2 mm,10 μm）with three groups of mobile phase at pH 3.0.Five methods were established based on the above columns,mobile phases and gradients,and their resolution of over-sulfated chondroitin sulfate and dermatan sulfate were compared.Results： Method E was the optimized method with the greatest resolution and theoretical plates,the limit of detection（LOD）and limit of quantitation（LOQ）for the over-sulfated chondroitin sulfate in heparin sodium were 0.025% and 0.03%,respectively.The LOD and LOQ for dermatan sulfate were 0.03% and 0.05%,respectively.The average recoveries of over-sulfated chondroitin sulfate and dermatan sulfate were 102.5% and 96.0%,respectively.Conclusion： This method is more accurate,sensitive,robust and reproducible compared with the existing pharmacopoeia method of heparin sodium products.