超高效液相色谱-串联质谱法测定螺旋藻多糖的单糖组成

建立了同时测定螺旋藻多糖水解产物中鼠李糖、木糖、阿拉伯糖、果糖、甘露糖、葡萄糖、半乳糖、甘露醇、核糖、岩藻糖、葡萄糖醛酸、半乳糖醛酸12种糖类化合物的超高效液相色谱-串联质谱分析方法。螺旋藻样品经超声波辅助提取,用三氟乙酸水解,经Waters Acquity BEH Amide色谱柱(100 mm×2.1 mm,1.7μm)分离,以10mmol/L甲酸铵和10 mmol/L甲酸铵-乙腈为流动相,在电喷雾电离源负离子(ESI-)模式下,用多反应监测(MRM)模式检测。结果表明,12种糖类化合物的定量限为0.005~0.15 mg/kg,线性范围为0.05~5 mg/L。按照样品中每种糖本底含量的50%、100%、150%进行添加,回收率为80.21%~121.6%。应用该方法对螺旋藻样品进行分析,结果发现:大部分样品都能检测到岩藻糖、半乳糖、阿拉伯糖、鼠李糖、葡萄糖、果糖、木糖、核糖,含量在0.3~889.4 mg/g之间。此外,测定的15个样品中岩藻糖、半乳糖、阿拉伯糖、鼠李糖、葡萄糖、果糖、木糖、核糖是共有组分,含量差异较大,但在所有样品中均未检测到甘露醇和甘露糖。该方法的建立可为阐明螺旋藻多糖的结构组成及其活性提供技术支撑及基础数据。

Determination of monosaccharide in Spirulina polysaccharide by ultra performance liquid chromatography-tandem mass spectrometry

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of 12 carbohydrates in hydrolysate of Spirulina including rhamnose, xylose, arabinose, fructose, mannose, glucose, galactose, mannitol, ribose, fucose, glucuronic acid and galacturonic acid. Samples were extracted with deionized water using ultrasonic-assisted extraction (UAE) and hydrolysis by trifluoroacetic acid (TFA). The hydrolysate was separated on a Waters Acquity BEH Aminde column (100 mm×2.1 mm, 1.7 μm) using 10 mmol/L ammonium formate/water -10 mmol/L ammonium formate/acetonitrile solution as the mobile phase. The electrospray ionization tandem quadrupole mass spectrometric analysis was carried out in the negative ion mode using multiple reaction monitoring (MRM). The limits of detection were 0.005-0.15 mg/kg and the linear ranges were 0.05-5 mg/L. The average recoveries were 80.21%-121.6% at the spiked levels of the carbohydrates contents of 50%, 100% and 150% found in samples. The method was applied to analyze the samples of Spirulina. Fucose, galactose, arabinose, rhamnose, glucose, fructose, xylose, and ribose were detected in most of the samples, and their contents were in the range of 0.3-889.4 mg/g. The type and content of carbohydrates that can be detected in the sample were significantly different. Common characteristic peaks were identified as fucose, galactose, arabinose, rhamnose, glucose, fructose, xylose, ribosethe. However, mannitol and mannose were not detected in all samples. This developed method could provide technical support and basic data for the study of structure and activity of polysaccharide from Spirulina.