Analytical Characterization Of Anthocyanins From Natural Products By Reverse-phase Liquid Chromatography-photodiode Array-electrospray Ionization-ion Trap-time Of Flight Mass Spectrometry

Abstract

Anthocyanins are known to be one of the most powerful phytochemical antioxidant and believed to have a positive influence on a variety of health conditions. Numerous studies continue on these compounds that are readily found in most plants. Red hybrid-tea rose petals and wild blueberries were used as model systems for optimizing the extraction and identification process of anthocyanins in plant material using high performance liquid chromatography (HPLC) and mass spectrometry (MS) without the use of authentic standards. A number of extraction parameters, including sample homogenization, solvent selection and acid type and amount, were varied to examine the effects on anthocyanin extraction from blueberries. Individual anthocyanins within the extract solution were separated by reverse phase HPLC and identified by elution order and tandem mass spectrometry. Further definitive identification of anthocyanins from other isobaric flavonoid species was demonstrated by multi-dimensional tandem mass spectrometry fragmentation (MSn). Cyanidin-3,5-diglucoside and pelargonidin-3,5-diglucoside were isolated from an extract of freeze dried rose petals via fractionation. These compounds were then structurally-characterized by multi-stage mass spectrometry fragmentation (up to MS6). Variations in the collision energy of the ion trap resulted in cleavage of the glycosides at the MS/MS, and MS3 stages; fragmentation of the anthocyanidin was recorded at the MS4 stage and beyond. Computational modeling was used to investigate the cross-ring cleavage fragment ions of cyanidin.