Title: Analysis of anthocyanins in pomegranates using LC/MS/MS Authors
Submitted to: American Society for Mass Spectrometry
Publication Type: Abstract Only
Publication Acceptance Date: March 31, 2011
Publication Date: June 9, 2011
Citation: Grimm, C.C., Lloyd, S.W., Stein, R.E., Beaulieu, J.C. 2011. Analysis of anthocyanins in pomegranates using LC/MS/MS. American Society for Mass Spectrometry. 1660. Technical Abstract: Anthocyanins consist of a 2-phenylbenzopyrylium moiety with a variety of polyhydroxyl, polymethoxyl, and glycosylated substituents. Their presence is responsible for the red to purplish colors associated with fresh fruits and berries. There is currently a strong interest in these compounds because of their potent anti-oxidant capabilities, and the associated health benefits. Because of the numerous possible structures of these compounds, they are generally hydrolyzed to form anthocyanidins, and then quantified using HPLC. In this study, we employ LC/MS and LC/MS/MS to look at the intact anthocyanins, and the possibility of quantifying them individually. Freshly pressed and commercial pomegranate juices were filtered through a 0.2 um filter. Juices were analyzed by LC/MS/MS, using an Agilent 1260 LC system, a Chip-cube interface, and a 6520 Q-TOF tandem mass spectrometer. A 43 mm analytical column packed with C18, and 5 µm beads with 80A pores was used. The capillary pump had a flow of 4 µl/min. The nano pump had a flow rate of 600 nL/min. An initial gradient (Solvent A-100% H2O, 0.1% Formic Acid, Solvent B- 90% ACN, 10% H2O and 0.1% Formic Acid) of 97% A, was changed to 30% Solvent A at 12 min, 0% at 13 min, 100% at 14 min, and 0% at 15 min. Juice samples were initially hydrolyzed to produce the anthocyanidins. LC/MS data indicated the presence of cyanidin, delphinidin, and pelargonidin. Their presence was confirmed by comparison of retention time and MS/MS spectra with known standards. No other anthocyanidins were detected. Detection of the 3-glycosides and 3,5-diglycosides was initially accomplished using auto MS/MS. For the 3-glucosides, the MS/MS spectra gave the parent ion and the neutral loss of a 162 Da to produce each of the three relative anthocyanidins from the respective parents. The MS/MS spectra of the 3,5-diglycosides was similar, with both the loss of 162 Da to produce the monoglycoside and an additional loss of 162 Da to produce the anthocyanidin. Evaluation of the MS spectra showed ions indicative of the possible presence of pentosides. Targeted MS/MS was employed to confirm the presence of a monopentaside and a diglycoside consisting of a pentose and a hexose. No anthocyanins containing two pentose moieties were detected. This is consistent with the reported presence of arabinose in pomegranates. Shoulders on the chromatographic peaks of the 3,5-diglyucosides of cyanidin and delphinidin suggest further isomerization. It is unclear if this results from the relative positions or hexose composition. Twelve different anthocyanins were observed in both commercial and fresh pomegranate juice employing LC/MS/MS.