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[摘要]
本研究建立了超高效液相色谱-串联质谱法测定蜂蜜中18种内源性酚酸、黄酮类物质检测方法。样品经盐酸水溶液提取,HLB固相萃取柱净化,采用Acquity UPLC HSS T3(2.1*100 mm,1.8 μm)色谱柱进行分离,以乙腈-10 mmol/L醋酸铵溶液为流动相进行梯度洗脱,电喷雾电离,负离子多反应监测模式检测,外标法定量。实验结果表明,所建立的方法线性范围宽,能适用于各类蜂蜜中酚酸、黄酮类物质的定量分析。各物质线性关系良好,相关系数(r2) ≥ 0.991;各分析物检出限在20~200 μg/kg之间,加标回收率范围为69.2%~94.1%,RSD为0.9%~9.5%。通过对150批次天然成熟蜂蜜、市售洋槐蜜及以不同比例(20%、40%、60%、80%)糖浆混合的洋槐蜜进行检测,发现不同蜜源蜂蜜中,酚酸、黄酮的种类与含量差异较大。其中,枣花蜜中阿魏酸平均含量最高,为550 μg/kg;洋槐蜜中芹菜素平均含量最高,含量为3910 μg/kg;咖啡酸在荆条蜜含量最高,为1721 μg/kg;糖浆掺假蜂蜜中目标物含量明显低于真正蜂蜜。该方法前处理操作简便、分析速度快、准确度高,可用于蜂蜜中多种内源性酚酸、黄酮类物质的同时测定。该方法为通过检测不同蜜源中酚酸及黄酮类物质含量建立蜂蜜指纹图谱,提供技术参考。
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[Abstract]
In this study, an analysis method using ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS) was developed for the determination of 18 endogenous phenolic acids and flavonoids in honey. The honey samples were extracted with an aqueous hydrogen chloride solution and purified with a Hydrophilic-Lipophilic-Balanced (HLB) column, and then separated on a high strength silica (HSS) T3 column and eluted with the mobile phase of acetonitrile-10 mmol/L ammonium acetate in a gradient mode, and analyzed by mass spectrometry in negative ion multiple reaction monitoring mode and using external standards. The obtained results showed that the established method exhibited a wide linear range and can be used for quantitative analysis of phenolic acids and flavonoids in various types of honey: A good linear relationship was found with a correlation coefficients (r2) not lower than 0.991. The detection limits for all the analytes ranged in 20~200 μg/kg, with the spike recoveries in the range of 69.2%~94.1% and relative standard deviations in the range of 0.9%~9.0%. It was found that the types and variations of phenolic acids and flavonoids in different honey sources differed largely, through analyses of 150 batches of natural mature honey, commercial acacia honey, and acacia honey blended with different proportions (20%, 40%, 60% and 80%) of syrup: the average content of ferulic acid in jujube nectar was the highest (550 μg/kg), the average content of apigenin in acacia honey was the highest (3910 μg/kg), and the content of caffeic acid in Vitex honey was the highest (1721 μg/kg), with the contents of the target compounds in the honey adulterated with syrup significantly lower than those in real honey. This analysis method exhibited advantages of simple pretreatment, high analysis speed and high accuracy, and can be used for simultaneous determination of various endogenous phenolic acids and flavonoids in various honeys. This method provides a technical reference for the establishment of honey fingerprints by comparing the contents of the phenolic acids and flavonoids in different samples.
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