LC-PDA-ESI-MS/MS PROFILING OF ANTHOCYANINS IN WILD BULGARIAN SMALL BERRY FRUITS

1. LC-PDA-ESI-MS/MS PROFILING OF ANTHOCYANINS IN WILD BULGARIAN SMALL BERRY FRUITS Ivayla Dincheva1, Ilian Badjakov1, Violeta Kondakova1, Patricia Dobson2, Gordon Mcdougall2, Derek Stewart2 1AgroBioInstitute, 8 DraganTsankov blvd., 1164, Sofia, Bulgaria 2The James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland UK Delphinidin-3-O-Galactoside Delphinidin-3-O-Glucoside Cyanidin-3-O-Glucoside Cyanidin-3-O-Galactoside Petunidin-3-O-Glucoside Petunidin-3-O-Glucoside Petunidin-3-O-Galactoside Cyanidin-3-O-Arabinoside Peonidin-3-O-Glucoside Malvidin-3-O-Arabinoside Peonidin-3-O-Galactoside Delphinidin-3-O-Arabinoside Malvidin-3-O-Glucoside Malvidin-3-O-Galactoside Peonidin-3-O-Arabinoside Abstract Wild berries have been associated with diverse health benefits, such as prevention of heart disease, hypertension, certain forms of cancer and other degenerative or age-related diseases. These beneficial health effects are due to their particularly high concentrations of natural antioxidants including anthocyanins, ascorbic acid, vitamins. HPLC-PDA method was used to determine anthocyanin content in wild small berries. Extracted anthocyanins were purified on a C18 solid phase extraction cartridge and characterized by HPLC-ESI-MS/MS. Anthocyanins were identified according to their retention times, elution order, and MS fragmentation pattern and by comparison with standards and published data. There were 15 dominant compounds in bilberry (VacciniummyrtillusL.), predominantly as monoglucosides of cyanidin, delphinidin, malvidin, peonidin and petunidin, 3 in lingonberry (Vacciniumvitis-idaeaL., mainly as monoglucosides of cyanidin, 12 in raspberry (RubusidaeusL.) and 8 in strawberry (FragariavescaL.), found as mono-, di- and triglucosides of cyanidin and pelargonidin. The purpose of this study was to use the complementary information obtained from HPLC analysis with PDA detector in combination with mass spectrometry to identify the structural characteristics of the conjugated forms of phenolic compounds as well as to quantify their content in wild small berries grown in the Rhodope Mountains. Sample preparation • Extraction with 0.1% (v/v) formic acid in 80% (v/v) MeOH; 30 min in ultrasonic bath A B • Centrifugation at 9000 rpm, 5 min 3 X Column: Synergy RP Hydro 80 Å 4µm (150 x 2 mm) • Evaporation to dryness in vacuum,t ≤ 35°C • EQUILIBRATION • 12 ml 0.1% (v/v) HCOOH inACN • 12 ml 0.1% (v/v) HCOOH in H2O • 1 - activation step • 2 - applied extract to the column • A - fraction sugars, organic and amino acids • B - fraction flavonoids 4°C Analyses of fractions ‘B’ @ positive ion mode, 520 nm • ELUTION • 12 ml 0.1% (v/v) HCOOH in H2O • 12 ml 0.1% (v/v) HCOOH inACN Strata C18-E (55µm, 70A), 2g/12ml Giga Tubes Cyanidin-3,5-O-diglucoside MW 611 Data analysis Results and discussion Detection of the most abundant anthocyanins in the bilberry extract by mass spectroscopy. The panels show the chromatographs of the presence of particular m/z values characteristic of the anthocyanins. The labels to the right of the panel represent the maximum intensity for each m/z value. R=Glu/Ara/Gal Cyanidin-3-O-sophroside MW 611 Identification of target metabolites This study describes a sensitive, fast, and accurate HPLC method to determine anthocyanins in berry fruits. The linearity, limits of detection, and limits of quantification were evaluated to determine the suitability of the method for analysis. To determine the appropriate calibration ranges for the target compounds, each sample was analyzed and compared to a solution containing known amounts of standards. As shown in Table 1, the anthocyanins exhibited linear peak area responses in their respective target ranges. The LODs for the anthocyanins were determined based on the concentration of the analyte that provides a peak height of 3x the measured baseline noise (S/N = 3), whereas the LOQs were determined as the concentration of the analyte that provides a peak height of 10x the measured baseline noise (S/N = 10). The LODs ranged from 0.20 μg/mL for Mal3Gal to 1.55 μg/mL for Dp3Glu and the LOQs from 0.75 μg/mL for Cy3Soph to 6.25 μg/mL for Dp3Glu. All samples were analyzed over three days to evaluate method precision. For the samples analyzed in this study, the intraday retention time RSDs ranged from 0.01% for Peo3Glu to 0.15% for Pet3Glu. Intraday peak area RSDs ranged from 0.75 for Cy3Glu to 5.95% for Pet3Glu. The between-day peak area RSDs ranged from 1.50% for Pet3Glu to 6.95% for Peo3Glu. Recovery studies were performed on all samples by spiking known amounts of the anthocyanins to determine method accuracy. Table 1 Data of Linearity, LOD and LOQ of Anthocyanins The discriminatory POWER of LS-ESI-MS/MS to differentiate between two compounds with similar MW PLS-DA scores of bilberry anthocyanins. The scores plot revealed a clustering of individuals (2009, 2010 and 2011) according to the type of habitat (sunny and shadow). Conclusions • Metabolite profiling of berry anthocyanins was achieved by LC-PDA-ESI-MS/MS. • Mass spectra provide more reliable discrimination of analytes with similar MW and identification the structural characteristics of the conjugated forms of phenolic compounds. • A combined metabolomic and statistical approach clearly functions as a high throughput method for establishing quantitative and qualitative chemical differences between species, with regard to the type of habitat (sunny and shadow). Acknowledgments: We acknowledge the support of the Bulgarian National Science Fund project № D002-334 and the EUBerry (EU FP7 KBBE-2010-4 grant agreement no. 265942).