高效阴离子交换色谱-脉冲安培检测法同时测定生物转化样品中的海藻糖、葡萄糖和麦芽糖

建立了高效阴离子交换色谱-脉冲安培电化学检测法同时测定生物转化样品中海藻糖、葡萄糖和麦芽糖的分析方法。选用CarboPac^TM10色谱柱(250 mm×2 mm)对分离条件进行优化,使用标准样品测定了线性范围和工作曲线,柱温为30 ℃,流速为0.30 mL/min,以氢氧化钠溶液和醋酸钠溶液为流动相进行梯度洗脱,脉冲安培法进行检测。研究结果表明,该方法可在15 min内实现海藻糖生物转化液中3种糖的快速定量分析。海藻糖、葡萄糖和麦芽糖峰面积与质量浓度的线性关系良好,检出限为0.010~0.100 mg/L。将此方法用于酶法制备海藻糖的检测,加标回收率为89.40%~103.2%。在生物转化样品中检测到海藻糖浓度为101.084 g/L,转化率达到了50.5%。该方法灵敏度高,简便快速,可应用于海藻糖制备样品中各种成分的分离和定量检测。

Simultaneous analysis of trehalose,glucose and maltose in biotransformation samples by high performance anion exchange chromatography with pulsed ampere detection

An analytical method for the determination of trehalose, maltose, and glucose in biotransformation samples was developed by using high performance anion exchange chromatography coupled with pulsed ampere detection (HPAEC-PAD). The analysis was performed on a CarboPac^TM10 column (250 mm×2 mm) with the gradient elution of NaOH-NaAc as the mobile phase. The column temperature was set at 30 ℃, the flow rate was 0.30 mL/min. The results showed that trehalose, maltose, and glucose in biotransformation system were completely separated and determined in 15 min. The linear ranges and the working curves were determined by using standard samples. The correlation coefficients of three kinds of carbohydrates were over 0.9998 . The detection limits (LODs) were 0.010-0.100 mg/L. Under the optimized separation conditions, the recoveries of saccharides in the transformation system at three different spiked levels ranged from 89.4% to 103.2%. In biotransformation system, 50 IU trehalose synthase were added into 200 g/L maltose for reaction of 8 h at 37 ℃, pH 8.0. Under the above conditions, the concentration of trehalose in biotransformation sample was 101.084 g/L, and the conversion rate of trehalose reached 50.5%. The method can be applied to determine the composition in the transformation system with the advantages of simplicity and convenience.