Determination of Endogenous Phenolic Acids and Flavonoids in Honey by UPLC/MS/MS
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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.
