Separation and characterisation of proanthocyanidins in Virginia type peanut skins by LC–MSn

Proanthocyanidins were extracted from peanut skins, an agricultural waste product of Virginia. Proanthocyanidin composition of single solvent and multistep extraction procedures of peanut skins were compared by HPLC–UV–vis absorbance. The multistep extraction procedure yielded more proanthocyanidin peaks and larger peak areas. Thus this extraction procedure was chosen for subsequent fractionation. Fractionation was performed by size exclusion (Toyopearl HW-40S) and normal phase (NP) (porous silica) high performance liquid chromatography (HPLC) before reversed phase (RP) electrospray ionisation liquid chromatography–mass spectrometry analysis (ESI–LC–MSn). Proanthocyanidin separation on the NP column was better overall, compared to size exclusion chromatography, and thus was chosen for characterisation by MS. Peanut skin procyanidins were found to separate in order of increasing molecular weight on the NP column. The ESI-MS generated positive and negative molecular-type ions and their dissociation product ions were used to identify specific proanthocyanidins and procyanidins. New proanthocyanidin dimeric and trimeric species were identified. These species consisted of one or two (epi)catechin units bound to luteolin or kaempferol. Monomers through nonamers of flavan-3-ols were identified by retention time and their multistep (MSn) dissociation pathway. Hexamers were identified as both singly and doubly charged negative ions, [M−zH]z−, z = 1 or 2. Species of greater than pentamers were tentatively identified due to their low ionisation efficiency and significant overlap of species both chromatographically and in the MS spectra.