HILIC-MS/MS法鉴别肝素和硫酸乙酰肝素

Identification of heparin and heparan sulfate by HILIC-MS/MS

目的建立亲水相互作用色谱-三重四极杆质谱法(HILIC-MS/MS)同时测定肝素及硫酸乙酰肝素经肝素酶Ⅰ、Ⅱ、Ⅲ酶切后产生的双糖含量,为肝素及硫酸乙酰肝素的鉴别提供方法和数据参考。方法采用PC HILIC色谱柱(4.6 mm×250 mm,5μm),以10 mmol/L乙酸铵-水溶液(A)、10 mmol/L乙酸铵-90%乙腈(B)为流动相梯度洗脱,流速0.9 mL/min,柱温40℃,进样量10μL,采用电喷雾电离源、负离子多反应检测模式对肝素及硫酸乙酰肝素酶切后产生的8种双糖进行定量,比较二者的差异。结果所测成分在测定摩尔浓度范围内线性关系良好(r2>0.9960),平均加样回收率为90.34%~99.62%,RSD为4.7%~6.7%,样品测定结果显示肝素与硫酸乙酰肝素双糖组成及比例差异明显。肝素中占比最大的双糖为ΔUA2S-GlcNS6S(TriS),约69.8%;硫酸乙酰肝素中占比最大的双糖为ΔUA-GlcNAc(0S),约51.0%。结论该法灵敏度高、稳定性好,简单快捷、准确可靠,可作为肝素与硫酸乙酰肝素的鉴别方法之一。

Objective To establish a hydrophilic interaction chromatography-mass spectrometry(HILIC-MS/MS)method for simultaneous determination of 8 disaccharides in heparin and heparan sulfate after digestion by heparinaseⅠ,ⅡandⅢ,and to provide method and data references for the identification of heparin and heparan sulfate.Methods A PC HILIC column(4.6 mm×250 mm,5μm)was used and gradiently eluted with 10 mmol/L ammonium acetate aqueous solution(A)and 10 mmol/L ammonium-90%acetonitrile(B)at the flow rate of 0.9 mL/min,column temperature of 40℃and injection volume of 10μL.The contents of the 8 disaccharides of heparin and heparan sulfate produced by enzymatic digestion were determined by electrospray ionization source and multi reaction monitoring mode and were compared with each other.Results The linear relationships of the 8 components were good in the range of measured molar concentrations(r2>0.9960),and the average recovery were 90.34%-99.62%,RSD were 4.7%-6.7%.The results showed that the disaccharides composition and proportion of heparin and heparan sulfate were significantly different.In heparin,ΔUA2 S-GlcNS6 S(TriS)accounted for about 69.8%on average,while in heparan sulfate,ΔUA-GlcNAc(0 S)accounted for about 51.0%.Conclusion The method established in this paper is simple,fast,accurate and reliable with high sensitivity,strong specificity,good stability,and can be used as one of the determination and identification methods for heparin and heparan sulfate.