HPLC-DAD methodology for the quantification of organic acids, furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo-diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250 4.6 mm, 5mm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan-3-ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans-resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n55) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rose ´ and fortified wines.

A fast method using a new hydrophilic–lipophilic balanced sorbent in combination with ultra-high performance liquid chromatography for quantification of significant bioactive metabolites in wines

This manuscript describes the development and validation of an ultra-fast, efficient, and high throughput analytical method based on ultra-high performance liquid chromatography (UHPLC) equipped with a photodiode array (PDA) detection system, for the simultaneous analysis of fifteen bioactive metabolites: gallic acid, protocatechuic acid, (−)-catechin, gentisic acid, (−)-epicatechin, syringic acid, p-coumaric acid, ferulic acid, m-coumaric acid, rutin, trans-resveratrol, myricetin, quercetin, cinnamic acid and kaempferol, in wines. A 50-mm column packed with 1.7-μm particles operating at elevated pressure (UHPLC strategy) was selected to attain ultra-fast analysis and highly efficient separations. In order to reduce the complexity of wine extract and improve the recovery efficiency, a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a new macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis™ HLB), was performed prior to UHPLC–PDA analysis. The calibration curves of bioactive metabolites showed good linearity within the established range. Limits of detection (LOD) and quantification (LOQ) ranged from 0.006 μg mL−1 to 0.58 μg mL−1, and from 0.019 μg mL−1 to 1.94 μg mL−1, for gallic and gentisic acids, respectively. The average recoveries ± SD for the three levels of concentration tested (n = 9) in red and white wines were, respectively, 89 ± 3% and 90 ± 2%. The repeatability expressed as relative standard deviation (RSD) was below 10% for all the metabolites assayed. The validated method was then applied to red and white wines from different geographical origins (Azores, Canary and Madeira Islands). The most abundant component in the analysed red wines was (−)-epicatechin followed by (−)-catechin and rutin, whereas in white wines syringic and p-coumaric acids were found the major phenolic metabolites. The method was completely validated, providing a sensitive analysis for bioactive phenolic metabolites detection and showing satisfactory data for all the parameters tested. Moreover, was revealed as an ultra-fast approach allowing the separation of the fifteen bioactive metabolites investigated with high resolution power within 5 min.

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.