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目的建立玻璃酸钠高效液相色谱测定法。方法用玻璃酸酶(来源于Streptococcus zooepidemicus)特异性水解玻璃酸钠生成不饱和二糖,反应体系为0.3 mol.L-1的醋酸钠溶液(pH 6.0),37℃水浴4 h,酶解液用流动相稀释后进样。色谱柱为Phenomenex Luna NH2(4.6 mm×250 mm,5μm);流动相为0.4 mol.L-1的磷酸二氢钠溶液;检测波长为232 nm。结果线性范围0.01～0.1 mg.mL-1(r=0.999 8),精密度和重复性良好,平均回收率为100.4%,RSD为0.8%。测定结果不被样品中包括硫酸软骨素在内的其他成分干扰。结论该方法简便准确,专属性强,可用于玻璃酸钠原料和玻璃酸钠制剂的含量测定。
Objective To establish a sodium hyaluronate high performance liquid chromatographic method. Methods Unsaturated disaccharide was hydrolyzed by hyaluronidase (derived from Streptococcus zooepidemicus). The reaction system was 0.3 mol·L-1 sodium acetate solution (pH 6.0), 37 ℃ water bath for 4 h, Dilute with mobile phase injection. The chromatographic column was Phenomenex Luna NH2 (4.6 mm × 250 mm, 5 μm). The mobile phase was 0.4 mol·L-1 sodium dihydrogen phosphate solution. The detection wavelength was 232 nm. Results The linear range was 0.01 ~ 0.1 mg.mL-1 (r = 0.999 8). The precision and reproducibility were good with the average recovery of 100.4% and RSD of 0.8%. The result of the measurement is not disturbed by other components in the sample including chondroitin sulfate. Conclusion The method is simple, accurate and specific. It can be used to determine the content of sodium hyaluronate and sodium hyaluronate.