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Mycotoxins are secondary metabolites produced by fungi that can contaminate a wide range of food and feed commodities and that are harmful to humans for their poisonous and toxic effects. An increasing amount of data have been accumulated in the last years, showing that mycotoxins may also occur in modified forms originating by plant, fungi or animal metabolism or by food processing. In particular, this modified forms may be produced via conjugation with sugars or other biological components (masked mycotoxins) or may occur as non extractable forms on account of strong interaction, association or binding with macromolecules in the food matrix (bound or hidden mycotoxins). Analytical methods have been set up in order to check for the occurrence of these forms and to evaluate their amount, in order to obtain reliable data for toxicity and exposure studies. In this paper hyphenated chromatographic methods for the determination and structural characterization of masked mycotoxins are reviewed, with a particular emphasis on liquid chromatography-(tandem) mass spectrometry as the most effective approach for their determination.

Head-space solid-phase microextraction (HS-SPME) coupled to gas-chromatography–mass spectrometry was developed and applied to obtain the volatile aromatic fingerprints of three typical Italian wines, Valpolicella, Amarone and Recioto, all produced in the restricted geographical area of Valpolicella (Veneto, Italy) with the same grape cultivars within the regulations of a rigid disciplinary of production. Differences between the three typologies are mainly linked to the different withering times to which grapes are subjected before vinification, which strongly influences the concentration and the development of volatile aroma compounds. A total of 22 different wines (7 Valpolicella, 10 Amarone and 5 Recioto) were characterised in terms of aromatic volatile profile with the aim to distinguish the different products and to evaluate the possibility to differentiate the same product from different brands. For the chemometric evaluation of the data one-way analysis of variance (ANOVA), principal component analysis (PCA) and hierarchical cluster analysis (HCA) were tested. All the chemometric tools employed allow to differentiate between the three products. More intriguing is the ability of the chemometric approach to differentiate between the same product (Amarone, Recioto) from different winery, thus showing the potential of this approach to characterize the brand-dependent typicality of wines, which is usually related to subtle technological differences which nevertheless have strong influences on the organoleptic characteristics of the products.

A LC/MS method for the simultaneous determination of both type A and type B trichothecenes by using an electrospray ionization (ESI) interface in the positive ionization mode with a single quadrupole analyzer is described. In order to enhance the ionization of both groups of trichothecenes, the sodium ion was used as cationization agent by adding sodium chloride to the eluent. All LC/MS parameters were optimized. The newly developed LC/ESI-MS method was applied to the analysis of a wheat reference material and cereal-based foods and feeds.

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 (R2or = 0.99), and applied to the analysis of spiked and naturally contaminated food samples.

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0-0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA-DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA-DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA-DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.

The enantiomeric separation of unmodified d , l -amino acids and Dns-amino acids in RP-HPLC by copper(II) complexes of terdentate diaminoamido ligands added to the eluent has been studied. The work is aimed at investigating whether a copper(II) complex with only one free equatorial position can still perform chiral discrimination of bidentate analytes such as unmodified and Dns-amino acids. The problem is approached by determining the nature and the stability of the initial copper(II) binary complexes and of the ternary complexes with amino acids in solution by potentiometry. Different chromatographic parameters were examined (pH, selector concentration, ionic strength, eluent polarity). All experimental data are consistent with a mechanism of chiral discrimination involving formation of diastereomeric complexes in which the amino acid coordinates at the free equatorial position and at the apical position of the initial complex which maintains its original chelate rings. It may still be considered a ligand exchange, implying that coordinated water molecules have been substituted by the analyte. The poor performance of the tricoordinated copper(II) complexes in the chiral discrimination of unmodified amino acids can be ascribed to the low stability of the mixed complexes and to their loose sterical requirements. With Dns-amino acids the most important feature seems to be the higher/lower affinity (match/mismatch) of the enantiomers for the selector adsorbed on the stationary phase.

Copper(II) complexes of l -amino acid amides added to the eluent in reversed-phase high-performance liquid chromatography are able to perform chiral discrimination of unmodified amino acids with high enantioselectivity. The mechanism is consistent with a ligand exchange between the binary initial copper(II) complex and the enantiomers. Evidence is provided that the exchange of the ligand is actually occurring during the chromatographic separation. The system involves a series of equilibria of exchange in the aqueous solution, in the stationary phase and between the two phases. Enantioselectivity is essentially due to the adsorption of the diastereomeric ternary species on the column, whereas the relative stabilities of the mixed complexes in the mobile phase seem to be negligible with respect to the overall discrimination process. The structural features of the initial copper complexes greatly affect the stereoselectivity of the process. The chromatographic parameters (pH, selector concentration, eluent polarity, ionic strength) are examined.

An HPLC system that allows the determination of the enantiomeric composition of complex mixtures of amino acids such as those occurring in biological fluids ( e.g. , serum, cerebrospinal fluid) and foods is described. d - and l -amino acids (including proline) can be determined. First, amino acid separation is achieved by means of an ion-exchange column by elution with a lithium chloride—lithium citrate buffer. Each peak corresponding to an individual amino acid can be switched to a reversed-phase column (C 18 ) and eluted with an aqueous solution containing chiral copper(II) complexes which perform chiral discrimination by a ligand-exchange mechanism. The method is very flexible as several chiral selectors and different types of detection ( e.g. , UV, fluorescence) can be used. Moreover, it avoids unnecessary overrunning of the chiral system with the whole mixture, by switching only the peaks under investigation. It is possible to evaluate d - amino acids up to a 0.1 % d to d + l ratio in the nanomolar range. Postcolumn derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and fluorimetric detection were utilized for proline and hydroxyproline and with o -phthaldehyde for the other amino acids.