Separation and Characterization of Phenolic Compounds from U.S. Pecans by Liquid Chromatography–Tandem Mass Spectrometry

Abstract

The phenolic acids and proanthocyanidins (PACs) of pecans possess bioactive properties, which might be useful in retarding the onset of and ameliorating the status of certain chronic disease states. There is a general lack of information in the literature regarding such compounds, especially the PACs. Crude phenolic extracts pooled from eight commercially significant cultivars were selected based on their relatively high antioxidant capacities. The pooled extracts were separated via Sephadex LH-20 column chromatography into five ethanolic low-molecular-weight (LMW) fractions and one acetonic high-molecular-weight (HMW) fraction. The preparations were then characterized using RP-HPLC-ESI-MS/MS and diol-phase HPLC-ESI-MS/MS in order to determine the key constituents present in the LMW and HMW fractions, respectively. As previously observed in pecan nutmeat, ellagic acid and (+)-catechin were found to be the major phenolics in the LMW fractions. The last eluting LMW fraction did not contain phenolic acids; rather it possessed PAC monomers and dimers. The HMW fraction comprised a majority of its PACs as dimers; yet, monomers, trimers, tetramers, pentamers, and hexamers were also separated and characterized.

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“…To determine the phenolic compounds which may be present in these LMW fractions, the fractions present in the extract were analyzed using RP-HPLC and the major constituents were identified and quantified, as presented by Robbins et al (2014). The results have been summarized in Table 1.…”

“…An example chromatogram at both 255 and 280 nm of the phenolic compounds isolated from a fraction III sample (i.e., part of the LMW sample) is highlighted in Figure 4. The phenolics were separated and identified by HPLC-ESI-MS/MS as reported by Robbins et al (2014).…”

“…Possible mechanisms proposed in the literature include changes in the expression of both COX-2 and iNOS enzymes, changes in the activation of NF-κB, and changes in the level of mRNA expression (Virgili et al, 1998; phenolic compounds in pecans may act in quenching key ROS which are involved in the activation of the NF-κB complex. Catechin and ellagic acid derivatives identified by RP-LC-MS/MS as described by Robbins et al (2014). e Ellagic acid contents include free ellagic acid and all other ellagic acid derivatives.…”

“…All quantities are expressed as mg compound/g tannin fraction. g PAC identification as described by Robbins et al (2014). h N/A means not applicable.…”

“…Spectral differences at key wavelengths (i.e., λ = 255 nm {ellagic acid and its derivatives}, 278 nm {phenolic acids, catechin, epicatechin}, 320 nm {phenolic acids of the trans-cinnamic acid family} and ~360 nm {flavonols}) were used as a guide to group the collected fractions into 5 pooled lots (Fractions I-V); pooling was based on observed similarities and differences in the spectra of the samples. Further details of the pooling process as well as a depiction of the spectral characteristics for the resultant fractions are given by Robbins, Ma, Wells, Greenspan, and Pegg (2014). After pooling, ethanol was evaporated using a Rotavapor with the water bath set at 45 °C.…”