Separation and characterization of phenolic compounds from dry-blanched peanut skins by liquid chromatography–electrospray ionization mass spectrometry

Highlights

• A large variety of phenolic compounds were identified in dry-blanched peanut skins (PS) by HPLC–MSⁿ.

• PS contain significantly more PACs compared to free phenolic compounds.

• p-Coumaroyl derivatives account for roughly 3/4 of the non-PAC phenolics found in PS.

• HILIC with fluorescent detection revealed the profile of the PACs found in PS.

• Our findings reported here greatly enrich the phenolics database of PS.

Abstract

A large variety of phenolic compounds, including phenolic acids (hydroxybenzoic acids, hydroxycinnamic acids, and their esters), stilbenes (trans-resveratrol and trans-piceatannol), flavan-3-ols (e.g., (−)-epicatechin, (+)-catechin, and their polymers {the proanthocyanidins, PACs}), other flavonoids (e.g., isoflavones, flavanols, and flavones, etc.) and biflavonoids (e.g., morelloflavone), were identified in dry-blanched peanut skins (PS) by this study. High-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI–MSⁿ) was applied to separate and identify the phenolic constituents. Reversed-phase HPLC was employed to separate free phenolic compounds as well as PAC monomers, dimers, and trimers. PACs with a degree of polymerization (DP) of >4 were chromatographed via hydrophilic interaction liquid chromatography (HILIC). Tentative identification of the separated phenolics was based solely on molecular ions and MSⁿ fragmentation patterns acquired by ESI–MS in the negative-ion mode. The connection sequence of PAC oligomers (DP <5) could be deduced mainly through characteristic quinone methide (QM) cleavage ions. When the DP reached 6, only a proportion of the flavan-3-ols could be ascertained in the PACs because of the extremely complicated fragmentation patterns involved. The identification of free phenolic acids, stilbenes, and flavonoids was achieved by authentic commercial standards and also by published literature data. Quantification was performed based on peak areas of the UV (free phenolic compounds) or fluorescence (PACs) signals from the HPLC chromatograms and calibration curves of commercial standards. Overall, PS contain significantly more PACs compared to free phenolic compounds.

Introduction

Phenolic compounds are ubiquitous in plants and have been classified as secondary metabolites [1]. Amongst the wide variety of phenolic compounds, phenolics of a dietary nature for humans typically include phenolic acids, flavonoids, and phenolic polymers (i.e., tannins) [2]. Phenolic acids (e.g., p-hydroxybenzoic acid and vanillic acid) can be present free; however, their common occurrence is in the form of methyl and ethyl esters, as well as glycosides being free and/or bound [1]. Hydroxycinnamic acids are mostly bound to cell wall polysaccharides through covalent linkages [1]. To illustrate, p-coumaric and ferulic acids are bound in graminaceous cell walls through ester-linkages between their carboxylic groups and arabinoxylans [3]. In plants, the common occurrences of hydroxycinnamic acids are as esters of hydroxyacids like quinic, shikimic, or tartaric acid [4], [5], [6]. Other conjugated forms of hydroxycinnamic acids include amides (e.g., with l-amino acids and peptides), esters of sugars, and glycosides [6]. The majority of flavonoids found in unpicked plants is generally in bound forms with one or more sugar residues attached [1], [5], [7]. As for flavan-3-ols (e.g., catechin, epicatechin, and gallocatechin) however, free monomers are also their common occurrence [1]. Mild heat treatment was found effective to cleave some of the covalent bonds of phenolic acids with insoluble polymers, and other cell wall components such as arabinoxylans in citrus peels thereby liberating low-molecular-weight antioxidant compounds from the repeating subunits of high-molecular-weight polymers [8]. These released compounds were further identified using gas chromatography–mass spectrometry (MS) analysis.

Peanut skins are the outer seed coat of the peanut cotyledon, and are often removed from peanuts before processing into products such as peanut butter. They are relatively high in proanthocyanidins (PACs). PACs are oligomers and polymers of flavan-3-ols, which are primarily linked by C4 → C8 bonds or less common C4 → C6 bonds (B-type), whereas A-type dimers contain an additional (C2 → O → C7) linkage. Despite the presence of some monomeric phenolic compounds, PACs comprise ∼17% by weight of peanut skins (PS) [9], and are the predominant phenolic component in PS [10]. Six A-type PAC dimers identified in PS [11] were found with activity to inhibit the inflammatory pathway mediated by hyaluronidase-induced release of histamine. A further study by Lou's group [12] led to the isolation of five PAC oligomers from the water-soluble fraction of PS with potential free-radical scavenging activity. Although A-type linkages are much more abundant, both A- and B-type PACs exist in PS. The degree of polymerization (DP) identified in PS was up to 12 bound by A-type linkages; whereas, B-type structures were detected with a DP of up to only six [13].

Because a greater polarity is associated with an increased DP, theoretically PACs can be fractionated by reversed-phase (RP) or normal-phase (NP) high-performance liquid chromatography (HPLC) in an order of ascending molecular mass [14]. However, the resolution of PACs is limited to tetramers on C₁₈ columns by RP-HPLC. The first successful separation of PACs was achieved on SiO₂ thin layer plates according to their DP [15]. Based on this thin layer chromatography method, a NP-HPLC gradient of CH₂Cl₂ and CH₃OH with traces of aqueous formic acid (1:1, v/v) was developed by Rigaud et al. [16], resulting in the separation of PACs up to a DP of five in a cacao bean extract. Hammerstone et al. [17] identified PAC decamers in cocoa by using a gradient of similar composition, but by replacing the formic acid with acetic acid. In the above cases, chlorinated solvents were employed in the mobile phase compositions; these solvents are environmentally unfriendly and can cause health problems from their exposure in the workplace. A better separation of PACs in cacao with a DP up to 14 was achieved by Kelm et al. [18] using a diol stationary phase with a relatively safe binary mobile phase of A (CH₃CN:CH₃COOH, 99:1, v/v) and B (CH₃OH:H₂O:CH₃COOH, 95:4:1, v/v/v).

The PAC molecules eluted via HPLC can be further ionized by an ion generator such as electrospray ionization (ESI), and these ions are subsequently separated according to their m/z ratio [14]. The HPLC–MSⁿ methods have been widely applied in the analysis of PACs in a variety of food matrices. Apropos PS: monomer (catechin and epicatechin), as well as A- and B-type PACs through dimers to tetramers were identified and quantified by Yu's group [10] with a RP-HPLC–ESI–MS system. Recently, Constanza et al. [19] reported the separation and identification of PACs through monomers to hexamers in PS using NP-HPLC–ESI–MS. Analyzing A-type PACs in PS, Appeldoorn et al. [20] identified 83 PAC species with DPs up to seven via a combination of NP-HPLC and RP-HPLC–ESI–MS². The fractions throughout NP-HPLC were further separated and characterized by RP-HPLC–MS². PS PACs up to nonamers were separated by NP-HPLC and characterized using RP-HPLC–ESI–MSⁿ in Sarnoski's study [21]. Montero et al. [22] described a comprehensive two-dimensional liquid chromatographic (LC × LC) technique, whereby both HILIC and RP-HPLC were applied as separation stages; this approach showed great potential in profiling the phenolic compounds of complex food systems.

According to Yu et al. [10], [23], the phenolics from PS are abundant not only in quantity but also in variety, which primarily includes phenolic acids (e.g., caffeic, chlorogenic, ferulic, and coumaric acids), flavan-3-ols (e.g., (epi)catechin, epigallocatechin, catechin gallate, and epicatechin gallate), stilbenes (e.g., trans-resveratrol), and PACs. However, only limited information can be found in the literature regarding phenolic profiles of PS from simple phenolic compounds to polymerized PACs using the combination technique of HPLC (both RP and HILIC) and MSⁿ. The objectives of the present study were as follows: (1) to assess the free phenolic compounds of the crude dry-blanched PS extracts using RP-HPLC–ESI–MSⁿ and (2) to assess the PACs profile of crude dry-blanched PS extracts using HILIC–ESI–MSⁿ.