New method for determination of (E)resveratrol in wine based on microextraction using packed sorbent and ultra-performance liquid chromatography

An ultra-fast and improved analytical methodology based on microextraction by packed sorbent (MEPS) combined with ultra-performance LC (UPLC) was developed and validated for determination of (E)-resveratrol in wines. Important factors affecting the performance of MEPS such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles, and sample volume were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (50–250mL) in one extraction cycle (extract–discard) and in a short time period (about 3 min for the entire sample preparation step). (E)-Resveratrol was eluted by 1 250mL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a highstrength silica HSS T3 analytical column (100 mm 2.1 mm, 1.8mm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, extraction yield, accuracy, and inter/intra-day precision, using a Madeira wine sample (ET) spiked with (E)-resveratrol at concentration levels ranging from 5 to 60mg/mL. Validation experiments revealed very good recovery rate of 9575.8% RSD, good linearity with r2 values 40.999 within the established concentration range, excellent repeatability (0.52%), and reproducibility (1.67%) values (expressed as RSD), thus demonstrating the robustness and accuracy of the MEPSC8/UPLC-photodiode array (PDA) method. The LOD of the method was 0.21mg/mL, whereas the LOQ was 0.68mg/mL. The validated methodology was applied to 30 commercial wines (24 red wines and six white wines) from different grape varieties, vintages, and regions. On the basis of the analytical validation, the MEPSC8/UPLC-PDA methodology shows to be an improved, sensitive, and ultra-fast approach for determination of (E)-resveratrol in wines with high resolving power within 6 min.

Development of a novel microextraction by packed sorbent-based approach followed by ultrahigh pressure liquid chromatography as a powerful technique for quantification phenolic constituents of biological interest in wines

A novel analytical approach, based on a miniaturized extraction technique, the microextraction by packed sorbent (MEPS), followed by ultrahigh pressure liquid chromatography (UHPLC) separation combined with a photodiode array (PDA) detection, has been developed and validated for the quantitative determination of sixteen biologically active phenolic constituents of wine. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (linearity, sensitivity, selectivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters on the MEPS performance such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). The wine bioactive phenolics were eluted by 250 μL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a HSS T3 analytical column (100 mm × 2.1 mm, 1.8 μm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method gave satisfactory results in terms of linearity with r2-values > 0.9986 within the established concentration range. The LOD varied from 85 ng mL−1 (ferulic acid) to 0.32 μg mL−1 ((+)-catechin), whereas the LOQ values from 0.028 μg mL−1 (ferulic acid) to 1.08 μg mL−1 ((+)-catechin). Typical recoveries ranged between 81.1 and 99.6% for red wines and between 77.1 and 99.3% for white wines, with relative standard deviations (RSD) no larger than 10%. The extraction yields of the MEPSC8/UHPLC–PDA methodology were found between 78.1 (syringic acid) and 99.6% (o-coumaric acid) for red wines and between 76.2 and 99.1% for white wines. The inter-day precision, expressed as the relative standard deviation (RSD%), varied between 0.2% (p-coumaric and o-coumaric acids) and 7.5% (gentisic acid) while the intra-day precision between 0.2% (o-coumaric and cinnamic acids) and 4.7% (gallic acid and (−)-epicatechin). On the basis of analytical validation, it is shown that the MEPSC8/UHPLC–PDA methodology proves to be an improved, reliable, and ultra-fast approach for wine bioactive phenolics analysis, because of its capability for determining simultaneously in a single chromatographic run several bioactive metabolites with high sensitivity, selectivity and resolving power within only 10 min. Preliminary studies have been carried out on 34 real whole wine samples, in order to assess the performance of the described procedure. The new approach offers decreased sample preparation and analysis time, and moreover is cheaper, more environmentally friendly and easier to perform as compared to traditional methodologies.

Biosystematics of the Genus Andryala L. (Asteraceae)

Andryala (Asteraceae: Cichorieae) is a little-known Mediterranean-Macaronesian genus whose taxonomy is much in need of revision. The aim of the present biosystematic study was to elucidate species relationships within this genus based on morphological and molecular data. In this study several taxa are recognised: 17 species, 14 subspecies, and 3 hybrids. Among these, 5 species are Macaronesian endemics (A. glandulosa, A. sparsiflora, A. crithmifolia Aiton, A. pinnatifida, and A. perezii), 4 species are Northwest African endemics (A. mogadorensis, A. maroccana, A. chevallieri, and A. nigricans) and one species is endemic to Romania (A. laevitomentosa). Historical background regarding taxonomic delimitation in the genus is addressed from Linnaean to present day concepts, as well as the origin of the name Andryala. The origin of Asteraceae and the systematic position of Andryala is shortly summarised. The morphological study was based on a bibliographic review and the revision of 1066 specimens of 13 herbaria as well as additional material collected during fieldwork. The variability of the morphological characters of the genus, including both vegetative taxonomic characters (root, stem, leaf and indumentum characters) and reproductive ones (inflorescence, floret, fruit and pappus characters), is assessed. Numerical analysis of the morphological data was performed using different similarity or dissimilarity measures and coefficients, as well as ordination and clustering methods. Results support the segregation of the recognised taxa and the congruence of the several analyses in the separation of the recognised taxa (using quantitative, binary or multi-state characters). The proposed taxonomy for Andryala includes a new infra-generic classification, new taxa and new combinations and ranks, typifications and diagnostic keys (one for the species and several for subspecies). For each taxon a list of synonyms, typification comments and a detailed description are provided, just as comments on taxonomy and nomenclature, and a brief discussion on karyology. Additionally, information on ecology and conservation status as well as on distribution and a list of studied material are also presented. Phylogenetic analyses based on different nuclear and chloroplast DNA markers, using Bayesian and maximum parsimony methods of inference, were performed. Results support three main lineages: separate ones for the relict species A. agardhii and A. laevitomentosa and a third including the majority of the Andryala species that underwent a relatively rapid and recent speciation. They also suggest a single colonization event of Madeira and the Canary Islands from the Mediterranean region, followed by insular speciation. Biogeography and speciation within the genus are briefly discussed, including a proposal for the centre of origin of the genus and possible dispersal routes.