Arnaldo Dossena

Applications of liquid chromatography–mass spectrometry for food analysis

HPLC-MS applications in the agrifood sector are among the fastest developing fields in science and industry. The present tutorial mini-review briefly describes this analytical methodology: HPLC, UHPLC, nano-HPLC on one hand, mass spectrometry (MS) and tandem mass spectrometry (MS/MS) on the other hand. Analytical results are grouped together based on the type of chemicals analyzed (lipids, carbohydrates, glycoproteins, vitamins, flavonoids, mycotoxins, pesticides, allergens and food additives). Results are also shown for various types of food (ham, cheese, milk, cereals, olive oil and wines). Although it is not an exhaustive list, it illustrates the main current directions of applications. Finally, one of the most important features, the characterization of food quality (including problems of authentication and adulteration) is discussed, together with a future outlook on future directions.

New reversed-phase liquid chromatographic method to detect aflatoxins in food and feed with cyclodextrins as fluorescence enhancers added to the eluent

The effect of succynil-beta-cyclodextrin (beta-CD-Su), dimethyl-beta-cyclodextrin (DIMEB) and beta-cyclodextrin (beta-CD) on the fluorescence of aflatoxins B1, B2, G1, G2 and M1 (AFB1, AFB2, AFG1, AFG2 and AFM1) was studied: beta-CD-Su promoted the largest fluorescence enhancement for AFB1 and AFM1 while DIMEB showed better results for AFG1 . On the basis of the fluorescence enhancement, a new RP-HPLC method for detecting aflatoxins B1, B2, G1, G2 and M1 was developed using cyclodextrins directly dissolved in the LC eluent. Aflatoxins B1, B2, G1 and G2 were resolved using a MICRA NPS ODS-1 column using methanol-water as mobile phase to which 6 x 10(-3) M beta-CD-Su or beta-CD were added. Chromatographic responses of AFB1 and AFG1 achieved using beta-CD dissolved in the mobile phase were enhanced, respectively, 8 and 12 times, and 10 and 15 times with beta-CD-Su. Detection limits lower than 0.3 microg/kg were achieved for all the four aflatoxins. Aflatoxin M1 was analysed using a Spherisorb S3 ODS-2 Narrow Bore column and methanol-water as mobile phase with added 2 x 10(-3) M beta-CD-Su. An area enhancement of 1.5 was detected for the toxin and the detection limit achieved under these analytical conditions was lower than 0.0005 microg/kg. Both methods were statistically validated showing a linear response for all the aflatoxins tested (R2 > or = 0.99), and applied to the analysis of spiked and naturally contaminated food samples.

Difficulties in fumonisin determination: the issue of hidden fumonisins

In this paper, the results obtained by five independent methods for the quantification of fumonisins B1, B2, and B3 in raw maize are reported. Five naturally contaminated maize samples and a reference material were analyzed in three different laboratories. Although each method was validated and common calibrants were used, a poor agreement about fumonisin contamination levels was obtained. In order to investigate the interactions among analyte and matrix leading to this lack of consistency, the occurrence of fumonisin derivatives was checked. Significant amounts of hidden fumonisins were detected for all the considered samples. Furthermore, the application of an in vitro digestion protocol to raw maize allowed for a higher recovery of native fumonisins, suggesting that the interaction occurring among analytes and matrix macromolecules is associative rather than covalent. Depending on the analytical method as well as the maize sample, only 37–68% of the total fumonisin concentrations were found to be extractable from the samples. These results are particularly impressive and significant in the case of the certified reference material, underlying the actual difficulties in ascertaining the trueness of a method for fumonisin determination, opening thus an important issue for risk assessment.

DNA Binding of AD-Lysine-Based Chiral PNA: Direction Control and Mismatch Recognition

Peptide nucleic acids (PNAs) are oligonucleotide analogues with a skeleton made up of N-(2-aminoethyl)glycine units; they bind to complementary DNA and RNA with high stability and specificity. In order to improve the binding specificity, solubility and uptake into cells, many modifications have been introduced, some concerning the introduction of stereogenic centres. With the aim of achieving a selective antiparallel binding with DNA, we report in this paper the synthesis and binding abilities of a chiral PNA decamer (H-GTAGATCACT-NH2) bearing three D-Lys-based monomers (a “chiral box”) in the middle of the strand. Indeed, the antiparallel PNA-DNA duplex showed a melting point of 43 °C (determined both by CD and UV spectroscopy), whereas the parallel PNA-DNA duplex failed to form, as shown by the absence of temperature dependence in the UV and CD spectra. Moreover, hybridization experiments carried out with antiparallel DNA strands bearing single mismatches showed that this PNA was excellent in discriminating between mismatched and matched targets. These results indicate that a high chiral constraint in the middle of a PNA sequence strongly affects the direction selectivity, i.e. the antiparallel/parallel preference in the DNA complexation. In particular, a “D-chiral box” favours a highly specific antiparallel DNA binding, thus allowing possible diagnostic applications for the screening of single-point mutations.

In Vitro Digestion Assay for Determination of Hidden Fumonisins in Maize

Hidden fumonisins have received great attention in the last years as they have been frequently found in maize products in addition to the free forms. Several papers have shown that interaction with macromolecular components such as protein and starch is at the base of the phenomenon: although the nature of the interaction (covalent or not) is still not clarified, the occurrence of hidden forms is generally revealed by the application of an alkaline hydrolysis procedure. In this study, an in vitro digestion model has been applied to raw maize to evaluate the possible release of hidden fumonisins under gastrointestinal conditions. Upon digestion of the food matrix, an increased amount of total detectable fumonisins was observed in comparison with the analysis on the nondigested matrix, an amount even higher than that calculated through the application of the hydrolysis procedure. Besides the analytical issues, our data have serious implications, since consumers may be exposed to a systematic higher risk than that estimated by conventional techniques.

The potential of enantioselective analysis as a quality control tool

Many food components are chiral: some are present as optically pure enantiomers, others are present in specific enantiomeric ratios. The enantiomeric ratio of some compounds, such as amino acids, can be affected by thermal treatments, fermentation and ageing. In the case of flavours, the enantiomeric ratio can be modified by the addition of synthetic racemic ingredients (adulteration). Enantiomers generally have different organoleptic properties, and may have different metabolic fates. The availability of highly sensitive chromatographic methods for chiral discrimination allows the determination of most enantiomers in foods. Thus, the enantiomeric ratio of food components is a reliable parameter to assess quality because it may be indicative of a technological process, contamination, adulteration, ageing and shelf life.

Free and bound fumonisins in gluten-free food products

In this work a multiresidual LC-ESI-MS/MS method for the simultaneous detection of free and bound fumonisins is described, which allowed for a very low LOD and a very good recovery for all the analytes. The method was applied to the determination of free and bound fumonisins in several gluten-free products from the Italian market. Free fumonisins were found to occur in 90% of the samples: the overall median value was below the EU legal limit for foods for human consumption (800 microg/kg). Nonetheless, fumonisins occurred in several samples at concentrations above the legal limit, reaching also very strong contamination levels (maximum concentration level: 3310 microg/kg). Anyway, considering the limited diet of people suffering of the celiac disease or allergic to other wheat proteins, the incidence of fumonisin contamination may be envisaged as problematic. Furthermore, bound fumonisins were found to be present in all the analysed samples at similar or even higher amounts than the free forms. In many cases the sum of free and bound fumonisins exceeded the EU legal limit for total fumonisins also for those samples characterized by a low contamination of free fumonisins, thus opening a new important task to be addressed for the risk assessment in this field.

Bis(L-amino acid amidato)copper(II) complexes as chiral eluents in the enantiomeric separation of D,L-dansylamino acids by reversed-phase high-performance liquid chromatography

Copper(II) complexes of L-amino acylamides (Phe, Val, Tyr, Ala) when added to the eluent (water-acetonitrile) in reversed-phase high-performance liquid chromatography (C18) are able to perform enantiomeric separation of dansylamino acids. The lipophilicity and bulk of the ligand greatly affect the stereoselectivity and the elution order of the enantiomers. The type and concentration of the copper complexes, pH and eluent polarity were examined in order to get some insights into the separation mechanism. This may be consistent with a ligand-exchange mechanism, probably occurring on the organic phase of the column, where the enantioselective complex is adsorbed. Mixtures of D,L-dansylamino acids were well separated by isocratic and gradient elution.

Solubilization and spectroscopic properties of α-chymotrypsin in cyclohexane

Abstract Using methyl-tryoctyl-ammonium chloride (which is soluble in cyclohexane and insoluble in water) it is possible to transport α-chymotrypsin in 20% yield from a water solution to a supernatant cyclohexane solution. The spectroscopic properties of the protein in the aprotic phase are investigated. On the basis of these spectroscopic data, it is argued that under certain conditions no extensive denaturation of the protein takes place in cyclohexane in the presence of the ammonium salt. The possible reason for this unexpected finding and its implications, are discussed.

Enantioselective sensing of amino acids by copper(II) complexes of phenylalanine-based fluorescent β-cyclodextrins

The synthesis and full characterisation of two modified cyclodextrins 6-deoxy-6- N-[N -(N 2 -dansylaminoethyl)R-(or S)-phenylalaninamide]- -cyclodextrin, containing a metal binding site and a dansyl fluorophore, are described. Both cyclodextrins were shown to form copper(II) complexes with fluorescence quenching. Addition of D- or L-amino acids to the copper(II) complexes induced a ‘switch on’ of the fluorescence which was enantioselective for Pro, Phe and Trp. The enantioselective fluorescence effect was used for the determination of the optical purity of proline.