高效液相色谱法测定玻璃酸钠的含量

Determination of Sodium Hyaluronate by HPLC

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 HPLC method for determination of sodium hyaluronate. METHODS Sodium hyalur- onate was specifically hydrolyze by hyaluronidase from Streptococcus zooepidemicus in 0. 3 mol· L-1 sodium acetate buffer （pH 6. 0） at 37 ℃ for 4 h. The mixture was diluted with mobile phase for analysis. The chromatography was performed on a Phenomenex Luna NH2 （4. 6 mm× 250 mm, 5 μm） column with 0. 4 mol·L-1 sodium dihydrogen phosphate as the mobile phase. The eluent was detected at 232 nm. RESULTS This method had excellent linearity in the range of 0. 01 - 0. 1 mg· mL- 1 （ n = 6,r = 0. 999 8 ） and good precision. The recovery was 100. 4% with RSD of 0. 8%. The determination results were not affected by other ingredients in the samples. CONCLUSION This method is convenient, accurate and specific for determination of sodium hyaluronate.