José M. Sendra

Gas chromatography coupled to mass spectrometry analysis of volatiles, sugars, organic acids and aminoacids in Valencia Late orange juice and reliability of the Automated Mass Spectral Deconvolution and Identification System for their automatic identification and quantification

Neutral volatiles and non-volatile polar compounds (sugars, organics acids and aminoacids) present in Valencia Late orange juice have been analysed by Gas Chromatography coupled to Mass Spectrometry (GC-MS). Before analysis, the neutral volatiles have been extracted by Headspace-Solid Phase Microextraction (HS-SPME), and the non-volatile polar compounds have been transformed to their corresponding volatile trimethylsilyl (TMS) derivatives. From the resulting raw GC-MS data files, the reliability of the Automated Mass Spectral Deconvolution and Identification System (AMDIS) to perform accurate identification and quantification of the compounds present in the sample has been tested. Hence, both raw GC-MS data files have been processed automatically by using AMDIS and manually by using Xcalibur™, the manufacturers data processing software for the GC-MS platform used. Results indicate that the reliability of AMDIS for accurate identification and quantification of the compounds present in the sample strongly depends on a number of operational settings, for both the MS and AMDIS, which must be optimized for the particular type of assayed sample. After optimization of these settings, AMDIS and Xcalibur™ yield practically the same results. A total of 85 volatiles and 22 polar compounds have been identified and quantified in Valencia Late orange juice.

LC-DAD-ESI/MSn Determination of Direct Condensation Flavanol-Anthocyanin Adducts in Pressure Extracted Pomegranate (Punica granatum L.) Juice

Pomegranate (Punica granatum L.) juice, obtained by pressure extraction of the whole fruit, has been analyzed for its flavanol-anthocyanin adduct content using reversed-phase liquid chromatography with diode array detection, coupled to mass spectrometry (ion trap) with electrospray ionization (HPLC-DAD-ESI/MS(n)), operating in positive ion mode. A total of 35 dimers have been detected, consisting of mono- and disubstituted hexoside derivatives of the adducts between the flavan-3-ols (epi)gallocatechin, (epi)catechin and (epi)afzelechin and the anthocyanidins delphinidin, cyanidin and pelargonidin. In addition, evidence is given for the presence of additional anthocyanin-flavanol adducts in this juice.

Reduction Kinetics of the Antiradical Probe 2,2-Diphenyl-1-picrylhydrazyl in Methanol and Acetonitrile by the Antiradical Activity of Protocatechuic Acid and Protocatechuic Acid Methyl Ester

This work evaluates the reduction kinetics of the antiradical probe 2,2-diphenyl-1-picrylhydrazyl (DPPH (*)) in methanol and acetonitrile by the antiradical activity of protocatechuic acid (3,4-dihydroxybenzoic acid, 1) and protocatechuic acid methyl ester ( 2). The reduction kinetics of DPPH (*) in both solvents by the antiradical activity of the p-catechol group in 2 is regular, that is, coincide with the proposed standard kinetic model for the reduction kinetics of DPPH (*) by the antiradical activity of an isolated p-catechol group. Therefore, the antiradical activity of 2 experimentally exhibits two rate-two stoichiometric constants in acetonitrile and three rate--three stoichiometric constants in methanol. In contrast, the reduction kinetics of DPPH (*) in both solvents by the antiradical activity of the p-catechol group in 1 is perturbed, that is, deviate from the proposed standard kinetic model. The deviations arise from the presence of the reactive carboxylic acid function which, in methanol, induces an additional reversible side reaction and, in acetonitrile, turns an irreversible reaction reversible, thus modifying the otherwise regular reduction kinetics of DPPH (*) by the antiradical activity of the p-catechol group in 1. On the other hand, the approximated theoretical kinetic equation that applies for those p-catechol groups whose reduction kinetics is regular and that experimentally exhibit three rate--three stoichiometric constants has been derived and used for fitting.

Identification of New Coloured Anthocyanin–Flavanol Adducts in Pressure-Extracted Pomegranate (Punica granatum L.) Juice by High-Performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry

Pomegranate (Punica granatum L.) juice, obtained by pressure extraction of the whole fruit, contains coloured flavanol–anthocyanin adducts (the flavanol occupies the upper part of the dimmer) from the direct condensation between anthocyanidins delphinidin, cyanidin and pelargonidin and flavan-3-ols (epi)gallocatechin, (epi)catechin and (epi)afzelechin. The presence of adducts between these same flavanols and anthocyanidins, but belonging to the coloured anthocyanin–flavanol adduct type (anthocyanin occupies the upper part of the dimmer) has been revealed by reversed-phase liquid chromatography coupled to tandem mass spectrometry (ion trap), with positive electrospray ionization (LC–ESI/MSn). These new adducts are isotopic with their corresponding counterparts (flavanol–anthocyanin adducts) and indistinguishable from them by comparison of the mass spectra from the MS2 of the isotopic parent ions. However, they can be distinguished by comparison of the mass spectra from the MS3 of their corresponding isotopic aglycons. Hence, the MS3 of the aglycon from a given flavanol–anthocyanin adduct always yields a mass spectrum containing five characteristic ions, the three with smaller m/z being only dependent on the anthocyanidin and the other two on both the anthocyanidin and the flavanol. In contrast, the mass spectrum from the MS3 of the aglycon of its counterpart anthocyanin–flavanol adduct gives only two of the above five characteristic ions, where the ion with smaller m/z only depends on the anthocyanidin and the other on both the anthocyanidin and the flavanol. Ten 3-hexoside derivatives of coloured anthocyanin–flavanol adducts were detected in pomegranate juice being reported for the first time this type of adducts from a natural source.

Detection of orange juice dilution by canonical correlation analysis

Abstract Canonical correlation analysis is used to detect orange juice dilutions masked by addition of citric acid and sugars. The values of 28 analytical characteristics (experimental variables) were determined in 105 pure orange juices, and canonical correlation analysis was applied to two groups of experimental variables (citric acid, sucrose, glucose, and fructose versus the other variables). The first pair of canonical correlation variables (one from each group) showed a correlation coefficient of 0.966. By dividing the whole set of 105 juices into reference sets (to compute predictive equations) and test sets (to check the efficiency of the equations), dilutions of 10, 20, and 30% were detected in, respectively, 28, 62, and 91% of the juices from the test sets.

Determination of the Depolymerisation Kinetics of Amylose, Amylopectin and Maltodextrin by Aspergillus niger Glucoamylase Using a 2-p-toluidinylnaphthalene-6-sulfonate/Flow-injection Analysis System with Fluorimetric Detection

This work describes the determination of depolymerisation kinetics of amylose, amylopectin and maltodextrin by Aspergillus niger glucoamylase using a flow-injection analysis system with fluorimetric detection and 2-p-toluidinylnaphthalene-2-sulfonate as the fluorescent probe. Experimental data corresponding to the time evolution of the concentration of detectable substrate were fitted to a single exponential decay curve in the case of amylose (linear substrate) and to a double exponential decay curve in the case of amylopectin and maltodextrin (ramified substrates). For all the substrates assayed, the depolymerisation rates at time zero correlated well with the initial substrate and enzyme concentrations through the Michaelis-Menten hyperbola. Therefore, this methodology allowed the determination of glucoamylase activity using any of these substrates. The determined value of the enzymic constant K m was lower for amylose than for amylopectin and maltodextrin, thus reflecting the higher difficulty of glucoamylase to hydrolyse the (1,6) when compared to the (1,4) linkages. In contrast, the values obtained for the rate constant k 3 were very similar for all the substrates assayed.

Determination of the antiradical activity and kinetics of pomegranate juice using 2,2-diphenylpicyrl-1-hydrazyl as the antiradical probe

Whole fruit pomegranate (Punica granatum L.) juice of the ‘Wonderful’ cultivar was characterized through the elucidation of its antiradical kinetics and activity using 2,2-diphenyl-1-picrylhydrazyl as the antiradical probe. Time-dependent concentration of 2,2-diphenyl-1-picrylhydrazyl during its reduction by the juice has been adjusted through a non-linear parametric fitting. Determined total antiradical activity was high, able to reduce 84.58 µmol/l of 2,2-diphenyl-1-picrylhydrazyl per concentration unit of juice (µl/ml), equivalent to a concentration of 42.29 mmol/l of ascorbic acid (or Trolox). Partial antiradical activities due to the fast-, medium- and slow-kinetics were 49.09, 18.16 and 17.33 µmol/l of reduced 2,2-diphenyl-1-picrylhydrazyl per concentration unit of juice (µl/ml), respectively. The corresponding rate constant for the fast-, medium- and slow-kinetics were κ1 = 6.03, κ2 = 0.169 and κ3 = 0.0094 (μl l)/(ml µmol min), respectively. This methodology allows characterization of samples through the accurate determination of the kinetics of their antiradical features, avoiding the use of empirical approximations that hinder the realistic comparison between extracts independently of their origin.

Time evolution of exposed hydrophobicity of water-soluble proteins during their depolymerisation by endo-proteases

During the depolymerisation of a water-soluble protein by an endo-protease, the exposed hydrophobicity of the substrate, that is the hydrophobicity that is accessible to hydrophobic probes, changes with the progress of the reaction. This work describes the depolymerisation of bovine serum albumin, α-casein and β-lactoglobulin using the proteases Alcalase, Flavourzyme, α-chymotrypsin, mercuripapain and trypsin. Time evolution of substrate hydrophobicity was monitored by a flow-injection analysis (FIA) system with fluorescence detection and an aqueous eluant containing p-toluidinylnaphthalene-6-sulfonate (2,6-TNS) as the fluorescent probe. In all cases, the time evolution of the substrate hydrophobicity was fitted using a derived mathematical function containing two adjustable rate constants and two constant parameters. This methodology allowed the determination of protease activities, as well as online monitoring of the depolymerisation process, when using water-soluble proteins as substrate.