Donatella Nardiello

Optimizing separation conditions for riboflavin, flavin mononucleotide and flavin adenine dinucleotide in capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the quantitative determination of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), using free solution capillary zone electrophoresis in uncoated fused-silica capillaries with laser-induced fluorescence (LIF) detection. Various factors influencing the separation and detection of flavin vitamers were investigated, including pH (5.5-10.5), concentration and nature of the run buffer (phosphate, borate and carbonate), applied voltage (15-30 kV), temperature (15-30 degrees C) and injection time. Optimal resolution and detection were obtained with a pH 9.8, 30 mM aqueous phosphate buffer at 15 degrees C and 30 kV of applied voltage. LIF detection was obtained with a He-Cd laser source using an excitation wavelength at 442 nm and lambda(em) > or = 515 nm. Riboflavin could be determined in the concentration ranges 0.5-350 microg/l with a rather low detection limit (LOD) down to 50 amol. The LODs of FAD and FMN were slightly higher, 300 and 350 amol, respectively. Combined with a simple clean-up procedure, the practical utility of this method is illustrated by the measurements of flavin derivates in foods and beverages, such as wines, milk, yoghurt and raw eggs.

Assessment of riboflavin and flavin content in common food samples by capillary electrophoresis with laser-induced fluorescence detection

To routinely assay the flavin contents in foodstuffs, a rapid and sensitive method was developed, in which the powerful separation capabilities of high-performance capillary electrophoresis (CE) were exploited. The method is based on a simple sample preparation, electrophoretic separation and laser induced fluorescence (LIF) detection. The average content of water-soluble riboflavin vitamers in raw natural products (i.e. vegetables, wheat flours and tomatoes) and bakers yeasts was evaluated without interferences from the sample matrices. Such an accurate and highly sensitive CE-LIF technique represents a significant improvement over previous analytical methods in terms of sensitivity, simplicity and efficiency. Indeed, it is well suited to satisfy the demands for accurate and sensitive detection with minimal sample preparation and clean-up.

Development of a new analytical method for the determination of sulfites in fresh meats and shrimps by ion-exchange chromatography with conductivity detection

An accurate and reliable analytical method, based on ion chromatography and suppressed conductivity detection, has been developed and validated for the quantitative determination of sulfites in fresh meats and shrimps. The chromatographic separation was accomplished by using an anion-exchange column eluted with sodium carbonate and sodium hydroxide. The optimized step-change elution, followed by column re-equilibration at the initial mobile phase composition, guaranteed a good resolution even toward endogenous interfering peaks, and an excellent retention time repeatability (1.1%, n=6). Good results in terms of sample extract stability, recovery efficiency were achieved with an extraction solvent mixture based on sodium hydroxide, fructose and EDTA. The method validation, performed by an in-house model according to Decision 657/2002/EC and Regulation 882/2004/EC, provided excellent results with respect to linearity (correlation coefficient up to 0.9998), limits of detection and quantification (2.7 and 8.2 mg kg(-1), respectively, expressed as SO(2)), expanded measurement uncertainty (below 10%), recovery values (ranging from 85% to 92%) and repeatability (down to 8%), demonstrating the conformity of the proposed method with the European directives. Finally, by major changes ruggedness studies, the method applicability to the quantitative analysis of cow hamburger, pork and horse sausage, and shrimps was demonstrated.

Validation of a confirmatory analytical method for the determination of aflatoxins B1, B2, G1 and G2 in foods and feed materials by HPLC with on-line photochemical derivatization and fluorescence detection

A sensitive and selective analytical method for the simultaneous separation and quantitative determination of aflatoxins B1, B2, G1 and G2 in foodstuffs and materials for feed has been validated. The method is based on high performance liquid chromatography with on-line post-column photochemical derivatization and fluorescence detection. The chromatographic separation of aflatoxins was accomplished using a C18 column eluted with an isocratic mobile phase consisting of water, methanol and acetonitrile. The sample preparation required a simple extraction of aflatoxins with MeOH/H2O (80:20, v/v) and a purification step by immunoaffinity column cleanup. The total analysis time, including sample preparation and chromatographic separation, did not exceed 40 min with a run time of 10 min. The on-line photochemical derivatization ensures better results in terms of simplicity, sensitivity and reproducibility with respect to chemical derivatization techniques, and provides an increase of the peak resolution and an extent of automation in comparison with the electrochemical ones. The procedure for the determination of aflatoxins in food samples and cereals for animal consumption was extensively validated following Regulation (EC) No. 882/2004. Detection limits in wheat bran samples of 0.08 µg kg−1 for AFB1, 0.02 µg kg−1 for AFB2, 0.16 µg kg−1 for AFG1 and 0.04 µg kg−1 for AFG2 were attained. The method allows high recovery with mean values ranging from 72 to 94% and it satisfies the necessary requirements for sensitivity, linearity, selectivity, precision and ruggedness, demonstrating the conformity of the method with provisions of Regulation (EC) No. 401/2006.

Quantitative determination of taurine in real samples by high-performance anion-exchange chromatography with integrated pulsed amperometric detection.

As taurine is a very important compound involved in a large number of metabolic processes, it is naturally present in the mammal tissues and is often deliberately added in some foods as a fortifying component. A detailed knowledge of taurine metabolic roles in biological systems can be obtained only if a sensitive, reliable and rapid analytical method is available. This article describes the successful application of high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD) for taurine determination in egg white and yolk samples, as well extracts of human serum and urine. Applications are shown for determination of taurine in soft drinks and pharmaceutical preparations where the taurine content was evaluated by standard additions. These results were achieved without prior derivatization of taurine.

Strategies in protein sequencing and characterization: multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry.

A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of number of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the analysis of peptides originating from the in-solution digestion of standard caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, number of experimental product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD experiments, high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymatic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously separated by two-dimensional gel electrophoresis.

Assessment of fumonisins B1 and B2 levels in commercial maize-based food products by liquid chromatography with fluorimetric detection and postcolumn chemical derivatization.

The occurrence of the fumonisins B(1) and B(2) in maize-based food products marketed in Italy was examined. A simply and reliable chromatographic method with fluorimetric detection and postcolumn o-phtalaldehyde derivatization was used for a monitoring of 100 samples (8 flours, 21 corn-meal, 16 snacks, 7 maize samples, 13 gluten-free products, and 35 corn-flakes) bought in local supermarkets during the years 2008 and 2009. The presence of both fumonisins B(1) and B(2), at a concentration higher than 15 μg/kg, was observed in all samples of corn-meal and maize-flour, in 75% of snacks, in 57% of maize samples, in 54% of gluten-free products, and in 29% of corn-flakes. A total of 7 samples including 4 corn-meals, 2 maize-flours, and 1 maize showed a value exceeding the maximum level fixed in the Regulation 1126/2007/EC; no positive sample was observed in corn-flakes, snacks, and gluten-free foods. Fumonisins contamination, on the whole range of maize-based food products analyzed, emphasizes the need of improve agricultural practices, and increase official control and monitoring studies.

Determination of deoxynivalenol and nivalenol by liquid chromatography and fluorimetric detection with on-line chemical post-column derivatization

A rapid, sensitive and selective analytical method was developed for the quantitative determination of deoxynivalenol (DON) and nivalenol (NIV) in cereals intended for human and animal consumption. The method, based on liquid chromatography and fluorescence detection, involves an automated 2 channel post-column derivatization, performed with sodium hydroxide, methyl acetoacetate and ammonium acetate. The chromatographic separation was accomplished using a C18 column eluted in isocratic mode with a mixture of 0.01% acetic acid and acetonitrile. Optimal fluorescence detection was obtained by an excitation and emission wavelength of 360 nm and 470 nm, respectively. The sample preparation required a rapid extraction of mycotoxins with water and a purification step by hydrophilic-lipophilic balance column clean-up. Under the optimized experimental conditions, a complete separation of DON and NIV was obtained in less than 20 min. The on-line post-column derivatization ensures excellent results in terms of simplicity and sensitivity, with limits of detection down to 0.014 mg/kg. The proposed method was extensively validated and the analytical performances of linearity (correlation coefficient of 0.9998), selectivity, precision (intra-day precision lower than 8%) and recovery (ranging from 89% to 101%) were evaluated, demonstrating the method feasibility in accurate confirmation analyses.

New Approach to Enrich Pasta with Polyphenols from Grape Marc

Food industry produces significant amount of waste that represents a problem for the sector. However, by-products are also promising sources of compounds which may be reused for their nutritional properties. The aim of this work is to exploit wine-making by-products, obtaining an extract by ultrasound-assisted extraction only using water as solvent. The characteristics of spaghetti enriched with grape marc were assessed and compared to control samples. In particular, total phenolic and flavonoids contents, the antioxidant activity, the cooking quality, and the sensory acceptability were evaluated at various steps of pasta production. The enriched spaghetti showed higher total phenolic and flavonoids contents and higher antioxidant activity than the control pasta. In addition, low cooking losses were found. In terms of sensory properties fortified pasta is acceptable as the traditional product, thus demonstrating that it is possible to exploit food waste to better satisfy consumer demand for healthy food products in a more sustainable perspective.

Determination of Fumonisins B 1 and B 2 in Maize Food Products by a New Analytical Method Based on High-Performance Liquid Chromatography and Fluorimetric Detection with Post-column Derivatization

A sensitive and selective analytical method for the quantitative determination of fumonisins B(1) (FB(1)) and B(2) (FB(2)) in maize-based foods for direct human consumption is described. The method, based on high-performance liquid chromatography and fluorescence detection, presents a rapid and automated online post-column derivatization, performed with o-phthalaldehyde and N,N-dimethyl-2-mercaptoethylamine (Thiofluor™). A complete separation of fumonisins is achieved in less than 13 min by using a C18 column and a gradient elution. Fumonisins are extracted from the sample with a mixture of water, acetonitrile, and methanol. The filtered extract is purified by immunoaffinity column and FB(1) and FB(2) are eluted with methanol. The method has been successfully validated, and performances comply with -criteria of the Regulation EC No 401/2006.