The occurrence of the fumonisins B1 and B2 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 B1 and B2, 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.

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.

A sensitive and accurate method for the determination of polyphenolic compounds in artichoke bract extracts and olive mill wastewaters by liquid chromatography coupled with pulsed amperometric detection at a glassy carbon working electrode was developed. Preliminary experiments were carried out by cyclic voltammetry to investigate the electrochemical behavior of polyphenols under different mobile phase compositions, and to test the detection and cleaning electrode potentials. Chromatographic separations were performed by using a core-shell C18column, eluted with acetic acid and acetonitrile, by combined concave-linear binary gradients. Under the optimized experimental conditions, a good column efficiency and peak symmetry were observed, also for stereo and positional isomeric compounds. The developed three-step potential waveform for pulsed amperometric detection was successfully applied for the sensitive chromatographic determination of polyphenols in artichoke extracts and olive mill wastewaters. Linearity, precision and sensitivity of the proposed method have been evaluated. A wide linear range of response (up to 20 mg/L) has been obtained for all the investigated compounds. Detection and quantification limits in the vegetable origin sample extracts were in the range 0.004–0.6 mg/L and 0.01–2 mg/L, respectively, while the injection-to-injection repeatability (n = 6) ranged from 5 to 13%. The obtained results confirmed the excellent sensitivity of the electrochemical detection, and its suitability for the determination of electroactive polyphenolic compounds at low concentration levels.

Effects of different packaging systems on water soluble oligopeptide fractions in fiordilatte cheese during its refrigerated storage are described. The degradation of the main protein fractions and the characterization of peptides arising from caseins were monitored by nanoliquid chromatography and electrospray ionization ion trap mass spectrometry (MS) coupled with a bioinformatics approach based on the scoring distribution and a post-database search validation. Microbiological and sensory properties of fiordilatte samples packaged under different conditions were also evaluated in order to find possible correlations between quality decay of cheese and peptide profile. The various packaging solutions differently responded to the degradation processes, thus allowing to identify the three systems that better preserved cheese quality: the two active coated samples packaged under MAP with and without brine and the sample in brine acidified at pH 5. As far as the peptide profile is concerned, a dynamic evolution of hydrophilic peptides was observed belonging almost exclusively to β-casein that could be regarded as the primary substrate of proteolysis in fiordilatte cheese. Characteristic trends with maximum and minimum values at different times were observed, throughout the refrigerated storage, leading to continuous changes in the peptide composition. A reduction in levels of β-casein peptides was observed in all the samples compared to the control, for which no specific packaging treatments were adopted.

Sistemi di derivatizzazione post-colonna per la determinazione di micotossine negli alimenti per uso umano e zootecnico, mediante cromatografia liquida a fase inversa e rivelazione fluorimetrica

The practice of adding adulterating substances (water, starch, sodium citrate, urea, sucrose, melamine, etc) in milk products in order to raise profits is worldwide employed. In addition, higher priced milk, coming from minor dairy species, such as goat and buffalo showing high nutritional and economical value, is often illegally integrated with the lower priced cow milk. The presence of species-specific proteins, different from those declared in label, may be a serious problem for people with allergies. The development of proper analytical methods is therefore essential to protect consumer benefits and product authenticity, as well as to detect economic frauds. Several analytical techniques, including capillary electrophoresis1, polymerase chain reaction2,

A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotranferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, -1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules.

A simple, rapid, and reliable multiresidue method, based on liquid chromatography (LC) and ultraviolet (UV)-diode array detection (DAD), is described for assaying ten sulfonamides (sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachloropyridazine, sulfamethoxazole, sulfaquinoxaline, and sulfadimethoxine) in feeds. The chromatographic separation is accomplished using a C18 column, eluted with a mobile phase consisting of acetate buffer, acetonitrile, and methanol. The sample preparation requires a simple extraction with chloroform/acetone and a purification step by solid-phase extraction. The analytical parameters of precision, detection and quantification limits, recovery, and ruggedness have been evaluated by a validation procedure following the European guidelines of Regulation 882/2004/EC and Decision 657/2002/EC.

In this work, the application of a new pulsed amperometric detection (PAD) waveform at a glassy carbon electrode, operating in typical chromatographic mobile phases, is proposed for the sensitive and reproducible determination of arylethanolaminic and phenolic moiety based compounds (e.g. betaagonists and polyphenols). Preliminary experiments by cyclic voltammetry were carried out to investigate the electrochemical behaviour and to select the detection and cleaning electrode potentials. The proposed potential-time profile was designed to prevent the carbon electrode fouling under repeated analyses, thus ensuring a reproducible and sensitive quantitative determination, without the need of any mechanical or chemical electrode cleaning procedure. The waveform electrochemical parameters, including detection and delay times, were optimized in terms of sensitivity, limit of detection and response stability. The optimized waveform allowed the sensitive and stable detection of model compounds, such as clenbuterol and caffeic acid, that showed detection limits of 0.1 mg L-1 and 14 mg L-1, quantification limits of 0.4 mg L-1 and 46 mg L-1, and linearity up to 100 mg L-1 (r = 0.9993) and 10 mg L-1 (r = 0.9998), respectively. Similar results were obtained for other compounds of the same classes, with precision values under repeatability conditions ranging from 3.0 to 5.9%. The proposed method can be then considered as an excellent alternative to the post-column detection of beta-agonists, phenols and polyphenols.

The problems and risks associated with mycotoxin contamination of foods and feedstuffs has led to the development of a variety of confirmatory analytical methods for their determination, based on chromatographic techniques and capillary electrophoresis. In this paper, an overview of the analytical methods developped in the last decade and based on post-column derivatization coupled with high-performance liquid chromatography and fluorescence detection is reported. A particular focus on the determination of aflatoxins (B1, B2, G1 and G2), fumonisins (B1 and B2) and trichotecenes deoxynivalenol (DON) and nivalenol (NIV) in cereals products for human and animal consumption is given. Important aspects as the optimization of the experimental conditions and validation of the analytical methods are also described.

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 characteriza- tion, 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.