Parallel enrichment of polyphenols and phytosterols from Pinot noir grape seeds with molecularly imprinted polymers and analysis by capillary high-performance liquid chromatography electrospray ionisation tandem mass spectrometry.

This investigation describes an integrated workflow for the parallel extraction and recovery of polyphenols and phytosterols from Pinot noir grape seeds. Using (E)-resveratrol and stigmasterol as exemplars, the approach employs two different molecular imprinted polymers in tandem for the extraction of these compounds and their subsequent analysis by capillary high-performance liquid chromatography (capHPLC) interfaced with electrospray ionisation tandem mass spectrometry (ESI MS/MS). Information on the selectivity of the solid-phase extraction processes was obtained through analysis of the binding behaviour of (E)-resveratrol- and stigmasterol-imprinted polymers using structurally similar polyphenols or phytosterols with the extent of binding determined from the capHPLC-ion trap ESI MS/MS data. This study documents with Pinot noir grape seed extracts and optimised solid-phase extraction protocols that the (E)-resveratrol-templated MIP enabled a very high recovery (99%) of the health-beneficial polyphenol (E)-resveratrol with co-purification of procyanidin and catechin/epicatechin. Further, the stigmasteryl-3-O-methacrylate-templated polymer resulted in high recovery (96%) of the phytosterol stigmasterol with co-purification of campesteryl glycoside. The results also demonstrate that rapid and high-resolution capHPLC-ESI MS/MS methods can be used as part of the work flow for selectivity optimisation and monitoring of the performance of MIPs intended for use in the solid-phase extraction of bioactive molecules with nutraceutical properties from agricultural waste streams.

Selectivity mapping of the binding sites of (E)-resveratrol imprinted polymers using structurally diverse polyphenolic compounds present in Pinot noir grape skins.

This investigation describes a general procedure for the selectivity mapping of molecularly imprinted polymers, using (E)-resveratrol-imprinted polymers as the exemplar, and polyphenolic compounds present in Pinot noir grape skin extracts as the test compounds. The procedure is based on the analysis of samples generated before and after solid-phase extraction of (E)-resveratrol and other polyphenols contained within the Pinot noir grape skins using (E)-resveratrol-imprinted polymers. Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionisation tandem mass spectrometry (ESI MS/MS) was then employed for compound analysis and identification. Under optimised solid-phase extraction conditions, the (E)-resveratrol-imprinted polymer showed high binding affinity and selectivity towards (E)-resveratrol, whilst no resveratrol was bound by the corresponding non-imprinted polymer. In addition, quercetin-3-O-glucuronide and a dimer of catechin-methyl-5-furfuraldehyde, which share some structural features with (E)-resveratrol, were also bound by the (E)-resveratrol-imprinted polymer. Polyphenols that were non-specifically retained by both the imprinted and non-imprinted polymer were (+)-catechin, a B-type procyanidin and (-)-epicatechin. The compounds that did not bind to the (E)-resveratrol molecularly imprinted polymer had at least one of the following molecular characteristics in comparison to the (E)-resveratrol template: (i) different spatial arrangements of their phenolic hydroxyl groups, (ii) less than three or more than four phenolic hydroxyl groups, or (iii) contained a bulky substituent moiety. The results show that capillary RP-HPLC in conjunction with ESI MS/MS represent very useful techniques for mapping the selectivity of the binding sites of imprinted polymer. Moreover, this procedure permits performance monitoring of the characteristics of molecularly imprinted polymers intended for solid-phase extraction of bioactive and nutraceutical molecules from diverse agricultural waste sources.

Recovery of ergosterol from the medicinal mushroom, Ganoderma tsugae var. Janniae, with a molecularly imprinted polymer derived from a cleavable monomer-template composite.

A semi-covalent imprinting strategy has been developed for the synthesis of molecularly-imprinted polymers specific for the fungal sterol, ergosterol, a biological precursor of vitamin D2. This imprinting approach involved a novel post-synthesis cleavable monomer-template composite, namely ergosteryl methacrylate, and resulted in the formation of an imprinted polymer that selectively and efficiently recognized ergosterol through non-covalent interactions. The derived molecularly-imprinted polymer and the corresponding non-imprinted polymer were systematically evaluated for their selectivity towards ergosterol via static and dynamic binding studies using various ergosteryl esters (e.g. ergosteryl-cinnamate, -ferulate, -coumarate, -ferulate acetate and -acetate, respectively) as competitors. Moreover, the binding capacity of the molecularly imprinted polymer for ergosterol was enhanced when the sample loading conditions involved the use of partially aqueous solvent mixtures, such as acetonitrile/water (9:1 (v/v) or 8:2 (v/v)). These attributes were exploited in a solid-phase extraction format, whereby ergosterol was obtained with excellent recoveries from an extract of the fruiting body powder of the medicinal fungus Ganoderma tsugae var. Janniae.

A comparison of covalent and non-covalent imprinting strategies for the synthesis of stigmasterol imprinted polymers.

Non-covalent and covalent imprinting strategies have been investigated for the synthesis of stigmasterol imprinted polymers. The synthesized molecularly imprinted polymers (MIPs) were then evaluated for their recognition and selectivity towards stigmasterol via static and dynamic batch-binding assays and their performance measured against control non-imprinted polymers (NIPs). MIPs prepared using the conventional non-covalent imprinting method displayed little to no binding affinity for stigmasterol under various conditions. In contrast, the application of a covalent imprinting approach using the novel post-synthetically cleavable monomer-template composite stigmasteryl-3-O-methacrylate resulted in the fabrication of a MIP that successfully recognized stigmasterol in both organic and partially aqueous environments. The affinity and selectivity of the covalently prepared MIP was enhanced when undertaken in a partially aqueous environment consisting of an acetonitrile/water (9:1, v/v) solvent mixture. These features have been exploited in a molecularly imprinted solid-phase extraction (MISPE) format, wherein the preferential retention of stigmasterol (with an imprint factor of 12) was demonstrated with 99% recovery in comparison to cholesterol (imprint factor of 6) and ergosterol (imprint factor of 4) while in the presence of several closely related steryl analogues.

Rapid solid-phase extraction and analysis of resveratrol and other polyphenols in red wine.

Red wine has long been credited as a good source of health-beneficial antioxidants, including the bioactive polyphenols catechin, quercetin, and (E)-resveratrol. In this paper, we report the application of reusable molecularly imprinted polymers (MIPs) for the selective and robust solid-phase extraction (SPE) and rapid analysis of (E)-resveratrol (LOD=8.87×10(-3) mg/L, LOQ=2.94×10(-2) mg/L), along with a range of other polyphenols from an Australian Pinot noir red wine. Optimization of the molecularly imprinted solid-phase extraction (MISPE) protocol resulted in the significant enrichment of (E)-resveratrol and several structurally related polyphenols. These secondary metabolites were subsequently identified by RP-HPLC and µLC-ESI ion trap MS/MS methods. The developed MISPE protocol employed low volumes of environmentally benign solvents selected according to the Green Chemistry principles, and resulted in the recovery of 99% of the total (E)-resveratrol present. These results further demonstrate the potential of generic protocols for the analysis of target compound with health beneficial properties within the food and nutraceutical industries using tailor-made MIPs.