Diego Airado-Rodríguez

Comprehensive two-dimensional liquid chromatography to quantify polyphenols in red wines.

A comprehensive two-dimensional liquid chromatography method has been applied for the quantification of polyphenols in red wines and compared to the most commonly employed conventional LC approach. Such methodology comprised the use of a microbore conventional HPLC column packed with totally porous particles in the first dimension and a partially porous column of conventional diameter in the second dimension. Even though a good number of applications in comprehensive LC have been reported, quantification experiments have been rarely described. To this regard, the advantages of comprehensive LC together with the employment of dedicated software capable of detecting and quantifying each peak from the 2D plot, have been taken into account for quantifying the most representative polyphenols in three different commercial Sicilian red wine samples. The optimized method has been validated in terms of linearity, sensitivity, detection and quantification limits. LODs as low as 0.02 ppm were obtained using the one-dimensional HPLC-DAD method, whereas values lower than 0.10 ppm were obtained by comprehensive LC. However, comprehensive LC allowed the quantification of a higher number of compounds with RSD lower than 10% thanks to its improved resolving power. The separation capabilities of comprehensive LC allowed the analysis of complex natural samples without any pre-treatment to effectively reduce the interferences coming from the matrix.

Assessment of the Quality Attributes of Cod Caviar Paste by Means of Front-Face Fluorescence Spectroscopy

This paper describes the fluorescent behavior of cod caviar paste, stored under different conditions, in terms of light exposure and concentration of oxygen in the headspace. Multivariate curve resolution was employed to decompose the overall fluorescence spectra into pure fluorescent components and calculate the relative concentrations of these components in the different samples. Profiles corresponding to protoporphyrin IX, photoprotoporphyrin, and fluorescent oxidation products were identified. Sensory evaluation, TBARS, and analysis of volatiles are typical methods employed in the routine analysis and quality control of such food. Successful calibration models were established between fluorescence and those routine methods. Correlation coefficients higher than 0.80 were found for 79% and higher than 0.90 for 50% of the assessed odors and flavors. For instance, R values of 0.94, and 0.96 were obtained for fresh and rancid flavors respectively, and 0.89 for TBARS. On the basis of these data, it can be argued that front-face fluorescence spectroscopy can substitute all of these expensive and tedious methodologies.

Effect of naturally occurring tetrapyrroles on photooxidation in cow's milk.

The objective of this work was to better understand the photosensitizing effect of riboflavin versus naturally occurring tetrapyrroles in cows milk. This was done by exposure of milk samples to blue light (400-500 nm), which is absorbed by riboflavin and tetrapyrroles, orange light (575-750 nm), which is absorbed by tetrapyrroles but not riboflavin, and white light, which contains the entire visible region. The milk was exposed to about 1.6 W/m(2) in 20 h, and two different light sources were tested: HMI lamp and fluorescent light tubes used for commercial display. Sensory analysis showed that wavelengths longer than 575 nm induced significantly more off-flavors than wavelengths shorter than 500 nm. By fluorescence spectroscopy it was observed that tetrapyrroles, in particular, chlorophyllic compounds, were degraded more by orange light than by blue and that the degree of degradation correlated closely with the formation of sensory off-flavors. The fluorescent agent Singlet Oxygen Sensor Green (SOSG) was used to monitor the formation of singlet oxygen under the different light exposure conditions, and the method verified that singlet oxygen was formed in large proportions in milk exposed to wavelengths longer than 575 nm, presumably with minor or no involvement of riboflavin. The results suggest that cholorophyllic compounds are responsible for a major part of photooxidation in milk. It is also suggested that β-carotene protects against photooxidation under blue light because it absorbs a major portion of the light below 500 nm and thereby reduces reactions with photosensitizers.

Sensitized synchronous fluorimetric determination of trans-resveratrol and trans-piceid in red wine based on their immobilization on nylon membranes.

It has been carried out the determination of trans-resveratrol and trans-piceid in red wine samples by using room temperature synchronous fluorescence, sensitized through their retention on nylon membranes, in front-face mode. These compounds are weakly fluorescent in solution but their retention allows using the native fluorescence of these compounds as analytical signal, due to the increase in the medium rigidity. To determine these compounds in red wine, a previous liquid-liquid extraction is necessary and in the case of trans-resveratrol it is also necessary a previous cleanup stage using C18 cartridges. Diethylether and ethyl acetate are the selected extractant solvents for trans-resvertarol and trans-piceid, respectively. The retention on nylon membranes was carried out by immersion of the membranes in solutions of these compounds. Variables involved in the retention and measurement processes were optimized, and the analytical figures of merit were obtained under optimal conditions. Ethanol:water 10:90 v:v and ethyl acetate were the solvents used for the retention of trans-resveratrol and trans-piceid, respectively and, for each case a immersion time of 300 and 600s was selected. Satisfactory linear relation between fluorescence intensity and concentration was found in the intervals 0.040 and 0.242mgL(-1) of trans-resvertarol and 0.009 and 0.288mgL(-1) of trans-piceid. Concentration of 1.08±0.21mgL(-1) for trans-resveratrol and 1.49±0.36mgL(-1) for trans-piceid were found in a wine sample obtained from a pool of commercial red wines.

Post-column on-line photochemical derivatization for the direct isocratic-LC-FLD analysis of resveratrol and piceid isomers in wine

Resveratrol is a stilbene produced by plants, e.g. grapes, in response to stress. Resveratrol is extracted during winemaking, being present in wine as 3-O-β-d-gluocoside (piceid) and as aglycone. Both, resveratrol and piceid exist in two isomeric forms, trans and cis. Resveratrol and piceid are weakly fluorescent in both of their isomeric forms, but highly fluorescent compounds are obtained when the original molecules are UV-irradiated. A chromatographic method with post-column on-line photoderivatization, has been developed for the analysis of resveratrol and piceid isomers. The four analytes are firstly separated in a C18 column (150mm×3.9mm i.d., 4μm) by isocratic elution, at 15°C, with a mobile phase consisting of a mixture acetonitrile:o-phosphoric acid (0.04%), 18:82, v:v, at 0.9mLmin(-1), and secondly they are on-line phototransformed into their fluorescent photoproducts in a 3m PTFE tube coiled around a 4W xenon lamp. The elution conditions have been chemometrically optimized by means of the experimental design and the response surface methodology. Linearity ranges from 0.10 to 1.50 and from 0.10 to 1.00μgmL(-1) and LOD around 0.001 and 0.01μgmL(-1) have been calculated for trans- and cis-isomers, respectively. The method has been satisfactorily applied to red and white wine samples by standard addition and external calibration, respectively.

From multispectral imaging of autofluorescence to chemical and sensory images of lipid oxidation in cod caviar paste.

The potential of multispectral imaging of autofluorescence to map sensory flavour properties and fluorophore concentrations in cod caviar paste has been investigated. Cod caviar paste was used as a case product and it was stored over time, under different headspace gas composition and light exposure conditions, to obtain a relevant span in lipid oxidation and sensory properties. Samples were divided in two sets, calibration and test sets, with 16 and 7 samples, respectively. A third set of samples was prepared with induced gradients in lipid oxidation and sensory properties by light exposure of certain parts of the sample surface. Front-face fluorescence emission images were obtained for excitation wavelength 382 nm at 11 different channels ranging from 400 to 700 nm. The analysis of the obtained sets of images was divided in two parts: First, in an effort to compress and extract relevant information, multivariate curve resolution was applied on the calibration set and three spectral components and their relative concentrations in each sample were obtained. The obtained profiles were employed to estimate the concentrations of each component in the images of the heterogeneous samples, giving chemical images of the distribution of fluorescent oxidation products, protoporphyrin IX and photoprotoporphyrin. Second, regression models for sensory attributes related to lipid oxidation were constructed based on the spectra of homogeneous samples from the calibration set. These models were successfully validated with the test set. The models were then applied for pixel-wise estimation of sensory flavours in the heterogeneous images, giving rise to sensory images. As far as we know this is the first time that sensory images of odour and flavour are obtained based on multispectral imaging.

Front-face fluorescence excitation-emission matrices in combination with three-way chemometrics for the discrimination and prediction of phenolic response to vineyard agronomic practices.

Phenolic extracts from cv Tempranillo grapes subjected to water stress and irrigation treatment, both of them with high and low crop load, were analyzed by front-face fluorescence. Excitation-emission matrices (EEMs) were analyzed by means of unsupervised parallel factor analysis (PARAFAC), PARAFAC supervised by linear discriminant analysis, and discriminant unfolded partial least-squares. All algorithms allowed to differentiate between water stress and irrigation grape samples when the fluorescence maxima region of catechin and epicatechin, and resveratrol was considered. A central composite design was employed for the calibration of catechin, epicatechin and resveratrol. Resveratrol was quantified by U-PLS in both, irrigated and water stressed samples, and levels between 3.46 ± 0.22 and 4.67 ± 0.08 μg mL-1 and 2.43 ± 0.60 and 3.03 ± 0.10 μg mL-1, respectively, were found. PARAFAC only allowed the determination of the sum of catechin plus epicatechin (R2 = 0.9397). The determination of total catechin plus epicatechin by means of PARAFAC was successfully validated by liquid chromatography.

Rapid and Nondestructive Determination of Aleurone Content in Pearling Fractions of Barley by Near-Infrared (NIR) and Fluorescence Spectroscopies

The aim of the present work was to develop and evaluate near-infrared (NIR) and fluorescence spectroscopies as rapid and potential online methods for determination of the amount of aleurone in pearling dust fractions of barley. Phytic acid was used as a marker for the aleurone layer. Different varieties of barley were pearled, and dust fractions were progressively taken out. Sample concentration of phytic acid varied in the range of 0.5-4.1 g/100 g, and highest concentrations were obtained in dust fractions for pearling degrees in the range of 15-25%. Regression models for phytic acid were developed with the same high correlations for NIR and fluorescence spectroscopies (R2 = 0.96) and prediction errors of ±0.16-0.18 g/100 g. The models performed well on a test set of pearling fractions from two other barley varieties. The techniques are rapid and nondestructive, which means that they can be used online in connection with industrial pearling systems.