流速补偿单糖外标法快速测定低聚糖的质量分布

Rapid determination of the mass distribution of oligosaccharide by external standard method with flow velocity compensation calibration monosaccharide

目的建立超高效液相色谱法结合蒸发光散射检测器(ultra performance liquid chromatography-evaporative light scattering detector, UPLC-ELSD)测定低聚糖不同聚合度质量分布的分析方法。方法以亲水作用机制来分离不同聚合度的低聚糖,再利用已知的不同聚合度糖标准品通过流速补偿建立蒸发光响应一致性方法,通过选择相对易得的单糖来建立标准曲线测定其他不同聚合度低聚糖的质量,最终得到不同聚合度低聚糖的质量分布。结果利用该方法分离测定了低聚果糖和低聚半乳糖样品的质量分布。采用果糖和半乳糖建立标准曲线,果糖在0.05~1.0 mg/mL范围相关系数(r^2)为0.9972,半乳糖在0.05~0.5 mg/mL范围相关系数(r^2)为0.9994。以0.25 mg/mL半乳糖和果糖标准溶液连续6次进样考察方法重复性,保留时间相对标准偏差(relative standard deviation, RSD)<0.1%,峰面积RSD<0.76%。低聚果糖的测定结果通过与蔗果三糖和蔗果四糖外标法结果的比对,最大偏差<2.5%,准确度较高。结论该方法快速、准确、成本低,可用于测定不同低聚糖的质量分布。

Objective To establish a method for the determination of mass distribution of the different degrees of polymerization for oligosaccharide by ultra performance liquid chromatography-evaporative light-scattering detector (UPLC-ELSD). Methods Oligosaccharide of different degrees of polymerization was separated by hydrophilic interaction mechanism. With the known sugar standards of different degrees of polymerization, evaporation light response uniformity was established through flow velocity compensation. Standard curves were first established by using accessible monosaccharides, then the mass distribution of the other oligosaccharide of different degrees of polymerization was determined by the standard curves. Finally, the mass distribution of oligosaccharide of different degrees of polymerization was obtained. Results By this method, the samples of fructooligosaccharides and galactooligosaccharides were separated and their mass distributions were determined. Through establishing standard curves by fructose and galactose, it was found that the correlation coefficient (r^2 ) of fructose in the range of 0.05-1.0 mg/mL was 0.9972, while the correlation coefficient (r^2 ) of galactose in the range of 0.05-0.5 mg/mL was 0.9994. The repetitiveness of the method was examined by 6 continuous sampling of 0.25 mg/mL standard solution of galactose and fructose, and the relative standard deviation (RSD) of retention time was <0.1% and the peak area RSD was <0.76%. The results of fructooligosaccharides were contrasted with the results of kestose and nystose determined by the external standard method, the maximum deviation was less than 2.5%, having relatively high accuracy. Conclusion The established method is fast, accurate, and cost-efficient, which is suitable for the determination of the mass distribution of oligosaccharide of different degrees of polymerization.