定量核磁共振氢谱法测定依诺肝素钠中多硫酸软骨素和硫酸皮肤素的含量

Determination of oversulfated chondroitine sulfate and dermatan sulfate in enoxaparin sodium by quantitative proton nuclear magnetic resonance

目的:建立测定依诺肝素钠中多硫酸软骨素(OSCS)和硫酸皮肤素(DS)含量的定量核磁共振氢谱(qHNMR)法。方法:采用Bruker AVANCE NEO 600 MHz核磁共振波谱仪采集核磁共振氢谱(1H NMR),90°脉冲,弛豫延迟时间为25 s,扫描次数为16次,测定温度为30℃,以2,2,3,3-d4-3-三甲基硅酸钠(TSP)作为内标物,对依诺肝素钠中的OSCS和DS进行定量分析。结果:OSCS、DS和TSP定量峰信号在1H NMR谱上分离度良好,OSCS和DS在各自线性范围内线性关系良好(r>0.9998),同时方法精密度(OSCS:1.4%;DS:1.5%)和重复性(OSCS:4.2%;DS:0.71%)好,测得依诺肝素钠产品中OSCS和DS含量分别为0.04%和0.06%~1.18%。结论:本文建立了测定依诺肝素钠中OSCS和DS含量的q HNMR法,简便快速,可作为依诺肝素钠中这2个杂质限度检查新的测定方法,也为其他低分子量肝素产品中OSCS、DS这2个杂质的测定提供参考。

Objective:To establish a quantitative proton nuclear magnetic resonance(qHNMR)method for the determination of the contents of oversulfated chondroitine sulfate(OSCS)and dermatan sulfate(DS)in enoxaparin sodium.Methods:The 1 H NMR spectra of enoxaparin sodium samples with OSCS and DS were collected by Bruker AVANCE NEO 600 MHz spectrometer with 90°pulse.The relaxation delay time was 25 s,the number of scanning was 16,and the experiment temperature was 30℃.2,2,3,3-d4-3-trimethyl sodium silicate(TSP)were selected as internal standards to determine the contents of OSCS and DS in enoxaparin sodium.Results:The quantitative peaks of OSCS,DS and TSP were separated on 1 H NMR spectrum.The method showed good linear relationship within the linear ranges(r>0.9998),and the precision(OSCS:1.4%;DS:1.5%)and the repeatability(OSCS:4.2%;DS:0.71%)were good,and the contents of OSCS and DS in enoxaparin sodium samples were 0.04%and 0.06%-1.18%,respectively.Conclusion:In this article,qHNMR method is established to determine the contents of OSCS and DS in enoxaparin sodium.The new method can be used for the determination of OSCS and DS impurities in enoxaparin sodium,and also provides a reference for the determination of two impurities in other low molecular weight heparin products.