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An evaluation of the grain functional components of Italian durum wheat cultivars was conducted. The raw material was obtained from the field trial performed in 2006-2007 at the Experimental Farm of the University of Bologna, (Bologna, Italy). The aim of this study was to define the phytochemical profile of ten varieties, comprised of old and modern durum wheat genotypes, including quantitative and qualitative phenolic and flavonoid content (free and bound forms). The results showed that mean values of total phenolic compound and total flavonoid content in old wheat varieties (878.2+/-19.0 micromol gallic acid equivalent/100g of grain and 122.6+/-25.4 micromol catechin equivalent/100g of grain, respectively) did not differ significantly from those detected in modern genotypes (865.9+/-128.9 micromol gallic acid equivalent/100g and 123.5+/-20.6 micromol catechin equivalent/100g, respectively). However, the HPLC-ESI-TOF-MS analysis highlighted remarkable differences between modern and old cultivars. The interpretation of the mass spectra allowed the identification of 70 phenolic compounds, including coumarins, phenolic acids, anthocyanins, flavones, isoflavones, proanthocyanidins, stilbenes and lignans. The free extracts of ancient wheat varieties showed the presence of a mean number of phenolic compounds and isomer forms (8.7+/-2.5 and 7.7+/-4.7 respectively) significantly higher than in modern genotypes (4.4+/-2.9 and 2.0+/-2.4, respectively). A similar trend was observed also for the bound phenolic fraction. Moreover, the phytochemical profiles showed the presence of unique phenolic compounds in both free and bound fractions of some of the investigated wheat genotypes. Results highlighted that investigated old wheat cultivars may offer unique nutraceutical values for their peculiar contents in bioactive phytochemicals, suggesting their uses into a wide range of regular and specialty products naturally enriched with health-promoting compounds.

The aim of this work was to develop a capillary electrophoresis-mass spectrometry (CE-MS) method to identify and quantify phenolic and other related polar compounds in walnut samples. The extraction capacity of several solvent mixtures of phenolic compounds from walnut by conventional solid-liquid extractions was tested, and CE and electrospray ionization MS parameters were optimized. The finalized procedure is able to determine many well-known phenolic compounds present in walnuts and provide relevant information about the presence of minor polar compounds. A new compound in walnut ((2E,4E)-8-hydroxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6-O-beta-d-glucopiranosyl ester, [M-H](-) 403.161m/z) with a structure similar to glansreginins was also identified. Phenolic compounds correspond to 14-28% of total polar compounds quantified. Aglycone and glycosylated ellagic acid represent the principal components and account for 64-75% of total phenols in walnuts. However, the sum of glansreginins A, B and (2E,4E)-8-hydroxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6'-O-beta-d-glucopiranosyl ester was in the range of 72-86% of total quantified compounds. In addition, this is the first time that separation by CE with detection by electrospray ionization time-of-flight MS has been applied to the analysis of phenolic and other polar compounds in walnut samples, providing results in less than 15min.

In the current work, a molecularly imprinted polymer (MIP) has been synthesised and used to enable the extraction of a naturally-occurring antioxidant from complex media. More specifically, we describe the first example of a caffeic acid (CA) MIP which has been synthesised in the form of well-defined polymer microspheres, and its use for the extraction of CA from fruit juice sample. The CA MIP was synthesised by precipitation polymerisation using 4-vinylpyridine as functional monomer, divinylbenzene-80 as crosslinker and acetonitrile:toluene (75/25, v/v) as porogen. The particle sizing and morphological characterisation of the polymers was carried out by means of scanning electron microscopy (narrow particle size distribution; ∼5 and 1.5 μm particle diameters for the MIP and NIP [non-imprinted polymer], respectively) and nitrogen sorption porosimetry (specific surface areas of 340 and 350 m(2)g(-1), and specific pore volumes of 0.17 and 0.19 cm(3)g(-1) for the MIP and NIP, respectively). The polymers were evaluated further by batch rebinding experiments, and from the derived isotherms their binding capacity and binding strength were determined (number of binding sites (N(K))=0.6 and 0.3 mmol g(-1) for the MIP and NIP, respectively, and apparent average adsorption constant (K(N))=10.0 and 1.6L mmol(-1) for the MIP and NIP, respectively). To evaluate the molecular recognition character of the MIP it was packed into a stainless steel column (50 mm × 4.6 mm i.d.) and evaluated as an HPLC-stationary phase. The mobile phase composition, flow rate, and the elution profile were then optimised in order to improve the peak shape without negatively affecting the imprinting factor (IF). Very interesting, promising properties were revealed. The imprinting factor (IF) under the optimised conditions was 11.9. Finally, when the imprinted LC column was used for the selective recognition of CA over eight related compounds, very good selectivity was obtained. This outcome enabled the direct extraction of CA in commercial apple juice samples with recoveries in excess of 81% and, rather significantly, without any need for a clean-up step prior to the extraction.

High-performance liquid chromatography with diode array and electrospray ionization mass spectrometric detection was used to carry out the comprehensive characterization of a lemon verbena extract with demonstrated antioxidant and antiinflammatory activity. Two different MS techniques have been coupled to HPLC: on one hand, time-of-flight mass spectrometry, and on the other hand, tandem mass spectrometry on an ion-trap. The use of a small particle size C18 column (1.8 microm) provided a great resolution and made possible the separation of several isomers. The UV-visible spectrophotometry was used to delimit the class of phenolic compound and the accurate mass measurements on time-of-flight spectrometer enabled to identify the compounds present in the extract. Finally, the fragmentation pattern obtained in MS-MS experiments confirmed the proposed structures. This procedure was able to determine many well-known phenolic compounds present in lemon verbena such as verbascoside and its derivatives, diglucuronide derivatives of apigenin and luteolin, and eukovoside. Also gardoside, verbasoside, cistanoside F, theveside, campneoside I, chrysoeriol-7-diglucuronide, forsythoside A and acacetin-7-diglucuronide were found for the first time in lemon verbena.

We have developed a direct method for the qualitative analysis of polyphenols in commercial organic fruit juices. The juices were diluted with water (50/50), filtered and directly injected. The analysis of phenolic compounds was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to photodiode array detection (DAD) and electrospray ionisation-Qq-time-of-flight mass spectrometry (ESI-Qq-TOF-MS). A unique gradient program has been optimized for the separation of several phenolic classes and the analysis time was only 5 min. The fruit juice samples were successfully analysed in positive and negative ionisation modes. In positive mode the anthocyanins were identified whereas the vast majority of polyphenols were identified using the negative ionisation mode. The sensitivity, together with mass accuracy and true isotopic pattern of the Qq-TOF-MS, allowed the identification of the phenolic compounds. Moreover, the advantage of the proposed method is the combined search of MS and MS/MS spectra, which improves the identification of compounds considerably, reducing ambiguities and false positive hits. Therefore the total fragmentation of the compound ion leading to the aglycone ion or other fragments was corroborated by MS-MS. The method was successfully employed to characterize diverse phenolic families in commercially available organic juices from four different fruits and consequently could be used in the future for the quantification purposes to compare different content of polyphenols in juices.

Eight antibiotics, chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline and rolitetracycline, were separated and quantified in Spanish honey extracts of different floral origin using a commercial RP-C18 HPLC column and two different on-line detectors (diode array and electrospray time-of-flight mass spectrometry (ESI-TOF-MS) systems). Operating a linear gradient at a flow of 2 ml min(-1) the HPLC separation of the eight antibiotics was obtained within 10 min with good peak symmetry and an acceptable resolution (2.1) for the critical band pair rolitetracycline and oxytetracycline. Values of the numbers of theoretical plates (N) were comprised between 2328 and 19448 while the limits of detection in honey were within 0.02-1.03 microg kg(-1) in the case of UV detection and 0.05-0.76 microg kg(-1) for ESI-TOF-MS detection (operating in negative mode). A recovery study was carried out by preparing some quality control samples at four levels of concentration (10, 25, 50 and 100 microg kg(-1)) and percentages between 72% and 98% were attained.

The use of calixarene compounds as selectivity modifiers and potassium bromide as electrolyte in capillary electrophoresis (CE) was studied. Calixarenes are macrocyclic oligomers having the shape of a conical vase whose inner cavity can accommodate various guest molecules. These compounds absorb in the ultraviolet region, with maxima at approximately 204 nm, and can be employed for the indirect determination of various types of compounds. The use of water soluble sulphonated calixarene as mobile phase in combination with different salts allowed us to develop electrophoretic methods for the determination of special compounds which are transparent in the UV region. The potential of this buffer was assessed by separating various groups of analytes including amino acids, biogenic amines and inorganic cations and anions. A system consisting of seven amino acids, used as model compounds, was chosen to optimize the buffer. The different instrumental variables affecting sensitivity and resolution of the mixture were carefully optimized.

El pdf del artículo es la versión pre-print.-- et al., In this contribution, the capabilities of pressurized liquid extraction (PLE) using food-grade solvents, such as water and ethanol, to obtain antioxidant extracts rich on polyphenolic compounds from olive leaves are studied. Different extraction conditions were tested, and the PLE obtained extracts were characterized in vitro according to their antioxidant capacity (using the DPPH radical scavenging and the TEAC assays) and total phenols amounts. The most active extracts were obtained with hot pressurized water at 200°C (EC50 18.6μg/mL) and liquid ethanol at 150°C (EC50 27.4μg/mL), attaining at these conditions high extraction yields, around 40 and 30%, respectively. The particular phenolic composition of the obtained extracts was characterized by LC-ESI-MS. Using this method, 25 different phenolic compounds could be tentatively identified, including phenolic acids, secoiridoids, hydroxycinnamic acid derivatives, flavonols and flavones. Among them, hydroxytyrosol, oleuropein and luteolin-glucoside were the main phenolic antioxidants and were quantified on the extracts together with other minor constituents, by means of a UPLC-MS/MS method. Results showed that using water as extracting agent, the amount of phenolic compounds increased with the extraction temperature, being hydroxytyrosol the main phenolic component on the water PLE olive leaves extracts, reaching up to 8.542mg/g dried extract. On the other hand, oleuropein was the main component on the extracts obtained with ethanol (6.156-2.819mg/g extract). Results described in this work demonstrate the good possibilities of using PLE as a useful technique for the valorization of by-products from the olive oil industry, such as olive leaves. © 2011 Elsevier B.V., This work was supported by AGL2008-05108-C03-01 (Ministerio de Educación y Ciencia) and CSD2007-00063 FUN-CFOOD (Programa CONSOLIDER-INGENIO 2010) projects. M.H. thanks the Spanish Science and Innovation Ministry (MICINN) for his “Ramón y Cajal” research contract. T.T.N. would like to thank the Asociación Hispano-Turca (AHIT) for the scholarship provided. M.P. thanks CSIC for her I3P fellowship.