Simultaneous LC-MS quantification of anthocyanins and non-anthocyanin phenolics from blueberries with widely divergent profiles and biological activities

Authors: GRACE, MARY - North Carolina State University; XIONG, JIA - North Carolina State University; ESPOSITO, DEBORA - North Carolina State University; Ehlenfeldt, Mark; LILA, MARY ANN - North Carolina State University

Submitted to: Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/22/2018
Publication Date: 10/28/2018
Citation: Grace, M.H., Xiong, J., Esposito, D., Ehlenfeldt, M.K., Lila, M. 2018. Simultaneous LC-MS quantification of anthocyanins and non-anthocyanin phenolics from blueberries with widely divergent profiles and biological activities. Food Chemistry. https://doi.org/10.1016/j.foodchem.2018.10.101.
DOI: https://doi.org/10.1016/j.foodchem.2018.10.101

Interpretive Summary: Speed and efficiency of tissue biochemical analysis is critical in comparing large and divergent plant populations. A rapid and accurate method utilizing laboratory techniques called liquid chromatography and mass spectrometry was developed and validated for the simultaneous detection and quantification of 38 components of blueberry fruit tissue in seven different blueberry plants with fruit color ranging from deep purple (blueberry species Vaccinium angustifolium) to various shades of pink (crosses of the blueberry species Vaccinium corymbosum, Vaccinium darrowii, and Vaccinium ashei). Samples were analyzed and results compared to those obtained from traditional methods of measurement. Our optimized method provided for easy identification and quantification of pigments and other health promoting compounds in blueberry samples; offering accurate results, shorter analysis time and use of chemical reagents relative to standard traditional methods of measurement. This information will be of use to researchers and food processors interested in nutritive value of blueberry fruit quality.

Technical Abstract: A rapid and accurate method based on liquid chromatography coupled with ion trap and time-of-flight mass spectrometry (LC-IT-TOF-MS) was developed and validated for the simultaneous detection and quantification of 22 anthocyanins, 4 flavan-3-ols, 7 flavonols, 4 phenolic acids, and resveratrol in 7 genotypes of blueberry with fruit color ranging from deep purple (Vaccinium angustifolium) to various shades of pink (crosses of V. corymbosum, V. darrowii, and V. ashei). Chromatographic separation was performed on a C18 column with a pore size of 12 nm. The conditions which led to the best base-line separation of anthocyanins from closely related structures was water with formic acid (0.5% v/v) (A) and methanol (B) as the eluent, with a flow rate of 0.6 mL/min, and a column temperature of 50 ºC. Anthocyanins and flavonols were determined in the positive ion mode, while flavan-3-ols, phenolic acids and resveratrol were measured in the negative ion mode, using an electrospray ionization interface. The method for quantification was accurate, reproducible, and the standard calibration curves were linear over a wide range of compound concentrations. The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.9959. The relative standard deviation (RSA) for intra- and inter-day precision over the concentration range of compounds was lower than 10%. In addition to phytochemical analysis, in vitro cellular antioxidant and anti-inflammatory bioassays were conducted on the 7 blueberry genotypes. The correlation between phytochemical composition and results of bioassays indicated that the anthocyanin class of phenolics has the highest contribution to the antioxidant and anti-inflammatory activities. The optimized LC-MS method allowed an easy and selective identification and quantification of anthocyanins and other phenolics in blueberry samples with divergent profiles; offering accurate results, shorter analysis time and reduced mobile phase volume with respect to conventional HPLC-DAD methods of quantitation.