葡萄糖直接发酵产2-酮基-L-古龙酸途径中代谢产物的HPLC分析

Analysis of Metabolites from Direct Fermentation of D-Glucose to 2-Keto-L-Gulonic Acid by HPLC

建立了利用高效液相色谱对葡萄糖发酵产2-酮基-L-古龙酸体系中的4种代谢产物D-葡萄糖酸、2-酮基-D-葡萄糖酸、2,5-二酮基-D-葡萄糖酸和2-酮基-L-古龙酸的定量测定方法,分析条件为:300mm×7.8 mm AminexHPX-87H柱,柱温30℃,流动相为50mmol/L硫酸,流速0.2mL/min,RID-10A示差检测器,以丙二酸为内标物.结果表明,4种物质分离较好.该方法对2-酮基-D-葡萄糖酸、2,5-二酮基-D-葡萄糖酸和2-酮基-L-古龙酸3种代谢物的检测平均误差分别为1.53%,0.89%和1.84%.

An HPLC-based method for quantitative determination of the 4 metabolites of gluconic acid （GlcA）, 2-keto-D-gluconic acid （2-KDG）, 2,5-diketo-D-gluconic acid （2,5-DKG） and 2-keto-L-gulonic acid （2-KLG） from direct fermentation of D-glucose to 2-KLG by using HPLC was established. The optimum separation of the 4 substances could be achieved under the conditions of using Aminex HPX-87H column （size 300 mm x7.8 ram） with the temperature of 30 ~C, sulfuric acid （50 mmol/L） as mobile phase at the flow rate of 0.2 mL/min, RID-10A detector, and malonic acid as internal standard. The analytical results show that the method has average analytical deviation of 1.53%, 0.89% and 1.84% on 2-KDG, 2,5-DKG and 2-KLG; respectively.