HPLC同时测定透明质酸发酵液中两种单糖含量的研究

Determination of Two Monosaccharides in Hyaluronic Acid Fermentation Broth by HPLC

目的建立一种高效液相色谱法定量检测透明质酸发酵液中的葡糖醛酸和乙酰氨基葡糖的含量。方法采用VenusilHILIC(250mm×4.6mm,5μm)色谱柱;以4mmol/L磷酸二氢钠溶液为流动相;柱温35℃;流速0.8ml/min;检测波长210nm。结果葡糖醛酸和乙酰氨基葡糖均在0.05~0.5mg/ml范围内有良好的线性关系(r=1.0000),平均回收率分别为97.1%和99.8%,相对标准偏差均为1.6%(n=9),检出限分别为20ppm和2ppm。结论该方法专属性强,回收率高,操作简便,适合于定量检测透明质酸发酵液中的葡糖醛酸和乙酰氨基葡糖含量,用以研究透明质酸发酵过程中单糖前体的转化情况。

Objective To establish an HPLC method for the determination of glucuronic acid and acetylglucosamine in hyaluronic acid fermentation broth simultaneously. Methods A Venusil HILIC column (250 mm×4.6 mm, 5 μm) was used with 4 mmol/L NaH2PO4 solution as mobile phase at 35 ℃, the flow rate was 0.8 ml/min, and the detection wavelength of the two monosaccharides was 210 nm. Results The linear ranges both were 0.05-0.5 mg/ml (r=1.0000) for glucuronic acid and acetylglucosamine, the average recoveries were 97.1 % and 99.8 %, with RSD of 1.6 % for both (n=9). The LOD were 20 ppm and 2 ppm for glucuronic acid and acetylglucosamine, respectively. Conclusion The method is simple, rapid, accurate and reproducible, and can be used for the quality control of monosaccharides in hyaluronic acid fermentation broth.