温和条件下柱前标记-高效液相色谱-质谱法测定枸杞多糖中单糖组成

Analysis of monosaccharide composition in polysaccharides from Lycium barbarum L. by precolumn labeling under mild conditions and high-performance liquid chromatography coupled with mass spectrometry

基于高效液相色谱-质谱联用分析技术(HPLC-MS),在温和的NH 3\5H 2O条件下,采用1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生化试剂成功地标记了果糖及多种醛单糖,同时提出了PMP标记果糖的衍生化机理。采用Kromasil-C18色谱柱(100 mm×4.6 mm,3.5μm)分离,梯度洗脱,选择离子模式检测,建立了8种常见单糖标记物的结构确认及含量测定的分析方法。该方法在一定质量浓度范围内具有良好的线性(相关系数>0.994 7),检出限为0.003~0.05 mg/L,定量限为0.01~0.15 mg/L。平均回收率为65.1%~116.2%,相对标准偏差≤10.2%(n=5)。自制4个产地枸杞多糖,对其单糖组成的分析结果表明,它们均由甘露糖、果糖、鼠李糖、半乳糖、葡萄糖、木糖、阿拉伯糖和脱氧核糖8种单糖组成,但各种单糖含量的分布有较大的差异。该法简便,灵敏度高,重复性好,可用于水热法水解枸杞多糖水解液中单糖标记物结构的确认及测定,对规范植物多糖的质量控制具有重要意义。

A method for the separation,identification,and determination of fructose and various aldehyde monosaccharides was established by precolumn labeling with 1-phenyl-3-methyl-5-pyrazolone(PMP)and high-performance liquid chromatography coupled with mass spectrometry(HPLC-MS).The separation was performed on a Kromasil-C18 column(100 mm×4.6 mm,3.5μm)with gradient elution.The detection was performed by electrospray ionization mass spectrometry in selected ion monitoring(SIM)mode.In this study,the derivatization mechanism of PMP-labeled fructose was proposed under mild NH 3\5H 2O conditions.The suggested method showed good linearity in the corresponding mass concentration ranges,with the correlation coefficients(r^2)>0.994 7.The limits of detection(LODs)and limits of quantification(LOQs)were in the ranges 0.003 to 0.05 mg/L and 0.01 to 0.15 mg/L,respectively.The recoveries in spiked Lycium barbarum L.samples were 65.1%to 116.2%,with relative standard deviations(RSDs)of less than 10.2%.By virtue of its simplicity,high sensitivity,and good repeatability,the method could be successfully applied to the analysis of the monosaccharide composition in polysaccharides of Lycium barbarum L.from four planting areas.Results showed that the isolated polysaccharides comprise mannose,fructose,rhamnose,galactose,glucose,xylose,arabinose,and ribose.The concentration distribution of various monosaccharides differed notably depending on the planted environmention.The proposed method is expected to be of great significance in standardizing the quality control of polysaccharides.