Biomedical subjects

A Dossena

Publications and source records attributed to A Dossena.

At least 19 recordsLinked to original sources

PubMed2004

Simultaneous detection of type A and type B trichothecenes in cereals by liquid chromatography-electrospray ionization mass spectrometry using NaCl as cationization agent.

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.

Chromatography, Liquid↗

PubMed2004

Simultaneous liquid chromatography-fluorescence analysis of type A and type B trichothecenes as fluorescent derivatives via reaction with coumarin-3-carbonyl chloride.

A method for the simultaneous LC-fluorescence detection (FLD) determination of eight trichothecenes A and B by pre-column derivatization with coumarin-3-carbonyl chloride, a highly fluorescent fluorophore, has been developed. The reaction conditions (temperature, reaction time, reactant ratios) were optimized to give a reproducible quantitative conversion. All derivatives were characterized by LC-MS. The chromatographic parameters were optimized (column, eluent) to give a very good separation of three type A (diacetoxyscirpenol, T-2 toxin, HT-2 toxin) and five type B trichothecenes [deoxynivalenol (DON), nivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol]. The best conditions were obtained on a narrow-bore C18 column with a water-methanol gradient. The detection limits (S/N = 3:1) in grain samples, with an injected volume of 5 microl, were 0.2-1 ng/g for all trichothecenes. These values are more than one order of magnitude lower than those of other LC-FLD and LC-MS methods and are similar to those obtained by GC-MS. The calibration curves were linear between 100 and 2500 ng/g. The method was successfully applied to the analysis of a certified wheat reference material, after solvent extraction and clean-up on a Mycosep column, obtaining a good recovery (89% for DON) and a high accuracy (z-score value: 0.67).

Calibration↗

PubMed2004

Reversed-phase liquid chromatographic method for the determination of ochratoxin A in wine.

In this paper, we propose a new, rapid, highly sensitive and reproducible RP-HPLC-FLD method for the detection of ochratoxin A (OTA) in wine, by directly injecting the liquid in the chromatographic system without any extraction or clean-up. An alkaline mobile phase (NH4Cl:CH-CN 85:15 (v/v), 20 mM, pH 9.8) was used to obtain a distinct fluorescence enhancement. This improvement allows to reach, without an immunoaffinity clean-up or concentration, a detection limit of 0.05 ng/ml, which is similar to those commonly obtained after immunoaffinity purification and acidic elution. The method was statistically validated and directly applied to a series of wine samples.

Chromatography, High Pressure Liquid↗

PubMed2003

Design and synthesis of fluorescent beta-cyclodextrins for the enantioselective sensing of alpha-amino acids.

Fluorescent monofunctionalized beta-cyclodextrins bearing a copper(II) binding side arm and a dansyl group (CD-NH-AA-CH(2)CH(2)NH-DNS) were designed as enantioselective sensors for unmodified alpha-amino acids. The side arm was derived from amino acid synthons (AA = L- and D-phenylalanine (1 and 2), L- and D-phenylglycine (3 and 4), L-proline (5), and L-cyclohexylglycine (6)) and was chosen in order to contain an amide, an amine, and a sulphonamide group. Enantioselectivity was evaluated by addition of copper(II) complexes of D- or L-valine and D- or L-proline. Chiral discrimination in the fluorescence response was observed in all cases, due to a ligand exchange process. The best conditions for these experiments were found to be the use of an excess (10:1) of the copper complex. The cyclodextrin 4 containing a D-phenylglycine unit was found to be poorly enantioselective, as found for 2, suggesting that the best design can be obtained by using L-amino acids. All L-amino acid containing cyclodextrins showed good enantioselectivities, some of which were higher than those already reported for 1. Other analytes related to amino acids were studied using cyclodextrins 1 and 3. Enantiomers of alpha,alpha-disubstituted amino acids, N-methylamino acids, and amino acid amides were found to be discriminated, while beta-phenylalanine and other molecules bearing a poor anchoring group at the alpha-carbon gave poor enantioselectivity. On the basis of the present data a model for the recognition process, based on the formation of ternary diastereomeric complexes, is proposed.

Amino Acids↗

PubMed2001

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.

Aflatoxins↗

PubMed2001

Recognition and strand displacement of DNA oligonucleotides by peptide nucleic acids (PNAs). High-performance ion-exchange chromatographic analysis.

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.

Base Sequence↗

PubMed2001

Chiral separation of amino acids by copper(II) complexes of tetradentate diaminodiamido-type ligands added to the eluent in reversed-phase high-performance liquid chromatography: a ligand exchange mechanism.

In this paper we report a study on the mechanism of the enantiomeric separation of unmodified D,L-amino acids in RP-HPLC by copper(II) complexes of two tetradentate diaminodiamido ligands, (S,S)-N,N'-bis(phenylalanyl)ethanediamine (PheNN-2) and (S,S)-N,N'-bis(methylphenylalanyl)ethanediamine (Me2PheNN-2), added to the eluent. The aim is to investigate whether and how a copper(II) complex with no free equatorial positions can perform chiral discrimination of bidentate analytes such as unmodified amino acids. The problem is approached in a systematic way by: (a) varying the different chromatographic parameters (pH, selector concentration, eluent polarity); (b) performing chiral separation with the selector adsorbed on the stationary phase; (c) studying the ternary complex formation of these ligands with D- and L-amino acids in solution by glass electrode potentiometry and electrospray ionization MS. All the experimental data are consistent with a mechanism of chiral recognition, based on ligand exchange, which involves as selectors the species [Cu2L2H(-2)]2+ and [CuLH(-2)] and proceeds by displacement of two binding sites from the equatorial positions, giving rise to the ternary species [CuLA]+ and [CuLH(-1) A]. The most important factor responsible for chiral discrimination seems to be the affinity of the diastereomeric ternary complexes for the stationary phase since no enantioselectivity is observed in solution.

Amino Acids↗

PubMed2001

Enantiomeric separation of hydroxy acids and carboxylic acids by diamino-beta-cyclodextrins (AB,AC,AD) in capillary electrophoresis.

Selectively modified 6,6'-dideoxy-6,6'-L-diamino-beta-cyclodextrins (AB, AC, AD) were successfully used as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids (in particular, phenoxyalkanoic acid herbicides) in capillary electrophoresis (CE). Chiral separations were obtained at a low selector concentration (1 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH. The different position of the charged groups on the upper rim greatly influenced the separation, accounting for electrostatic interactions between the protonated amino groups of the cyclodextrins (CDs) and the carboxylate of the selectands. The best enantiomeric separation of hydroxy acids was obtained with the AC regioisomer, whereas carboxylic acids were well resolved only by the AB regioisomer. A recognition model is proposed, based on two-dimensional nuclear magnetic resonance (2-D NMR) experiments, in which the orientation of the guest in the inclusion complex is determined by the electrostatic interactions between the selectand and the CD upper rim.

Carboxylic Acids↗

PubMed2000

Pyroglutamic acid in cheese: presence, origin, and correlation with ripening time of Grana Padano cheese.

Pyroglutamic acid is present in many cheese varieties and particularly in high amounts (0.5 g/100 g of cheese) in extensively ripened Italian cheeses (Grana Padano and Parmigiano Reggiano) that are produced with thermophilic lactic acid bacteria as starters. The mechanism of pyroglutamic acid formation in cheese seems to be mostly enzymatic, as demonstrated by the presence of only L-pyroglutamic acid enantiomer. Thermophilic lactobacilli are involved in pyroglutamic acid production, as suggested by the low pyroglutamic acid content found in Bagos, a ripened Italian mountain cheese produced without addition of starter. Because milk pasteurization did not influence the pyroglutamic acid content in the ripened Grana Padano cheese, the formation of pyroglutamic acid mainly depends on the whey starter microflora rather than that of raw milk. Pyroglutamic acid concentration is linearly correlated (R2 = 0.94) with the age of Grana Padano cheese.

Animals↗

PubMed1999

Histamine-modified cationic beta-cyclodextrins as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids by capillary electrophoresis.

The enantiomeric separation of alpha-hydroxy acids and carboxylic acids was successfully performed by using 6-deoxy-6-N-histamino-beta-cyclodextrin (CD-hm), a monosubstituted positively charged beta-cyclodextrin (beta-CD) bearing a histamine moiety linked to the C6 of a glucose unit in the upper CD rim via the amino group. Good results were obtained at a low selector concentration (1 mM). The number of positive charges on the upper rim may be modulated as a function of pH, because of the different pKa of the amino and the imidazolyl groups, and was found to affect both the enantioselectivity and resolution factors. With the analogous 6-deoxy-[4-(2-aminoethyl)imidazolyl]-beta-cyclodextrin (CD-mh) bearing the histamine moiety linked to the C6 via the imidazolyl group, very poor results were obtained, showing that the proximity of the positive charge to the cavity plays an important role in the enantiomeric recognition. The complexation mode was studied by electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance (2-D NMR) ROESY experiments: the recognition model is consistent with an inclusion complexation of the aromatic ring of the analyte within the CD cavity coupled to electrostatic interactions between the carboxylate and the protonated amino group of the cyclodextrin.

Carboxylic Acids↗

PubMed1997

Histamine-modified beta-cyclodextrins for the enantiomeric separation of dansyl-amino acids in capillary electrophoresis.

Two novel monosubstituted beta-cyclodextrins (CD) bearing the histamine moiety linked to the upper CD rim, either through the amino group or the imidazole nitrogen 1N, CD-hm and CD-mh, were successfully used as chiral selectors in capillary electrophoresis for the enantiomeric discrimination of dansyl (Dns)-amino acids. Good results were obtained by using low concentrations of the selectors (1-3 mM). The effect of pH on the chiral discrimination was studied in order to modulate the number and the position of the positive charges. By increasing the pH from 5 to 7.5, chiral discrimination decreased along with the deprotonation of the imidazolyl moiety. Inversion of the migration order was observed with the two CDs, depending on the relative position of the charged moieties on the upper rim. Ion pair interaction coupled to inclusion complexation seems to account for the discrimination process. The effects of the temperature, CD concentration and capillary length on chiral resolution were also examined.

Carbohydrate Sequence↗

PubMed1997

[Late awakening].

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Anesthesia↗

PubMed1996

Copper(II) complexes of N2-alkyl-(S)-amino acid amides as chiral selectors for dynamically coated chiral stationary phases in RP-HPLC.

Copper(II) complexes of N2-octyl-(S)-phenylalaninamide (Noc-Phe-NH2), N2-dodecyl-(S)-phenylalaninamide (Ndo-Phe-NH2), and N2-octyl-(S)-norleucinamide (Noc-NLeu-NH2), dynamically adsorbed on a reversed-phase C18 column, were able to perform the direct enantiomeric separation of unmodified amino acids, amino acid amides and esters, hydroxy acids, and dipeptides by elution with aqueous or mixed aqueous-organic solutions containing copper(II) sulphate or acetate. The role played by several parameters in the separation procedure was examined with the copper(II) complex of Noc-Phe-NH2 [concentration of the copper(II) ion in the eluent, pH and eluent polarity, amount of adsorbed selector]. The separation was shown to occur entirely on the stationary phase. The mechanism of chiral discrimination is discussed in terms of the chromatographic parameters and of the structure of the copper(II) complexes in solution and in the solid state. The chiral stationary phase maintained its separation ability for about 3 months. However, the column could be easily restored by recovering the selector with methanol and repeating the loading procedure.

Amides↗

PubMed1993

Diaminomethane dihydrochloride, a novel reagent for the synthesis of primary amides of amino acids and peptides from active esters.

Amide formation from acids, N-protected amino acids and peptides was achieved in an easy and convenient way by treating "active esters" such as succinimidyl or 4-nitrophenyl esters or acyl chlorides with diaminomethane dihydrochloride in dioxane in the presence of Et3N. Diaminomethane dihydrochloride behaves as a slow ammonia-releasing agent. The method is a good alternative to the use of concentrated aqueous ammonia; it avoids solubility problems and allows better control of the stoichiometry of the reaction and of the pH. It gives good yields and does not induce racemization. The mechanism of the reaction is discussed.

Amides↗

PubMed1992

Supramolecular selective transport of Ca2+/Na+ by dicarboxylic ligands across bulk liquid membranes.

Bulk liquid membrane transport of Ca2+ and Na+ ions was performed with dicarboxylic type ligands containing (S)-phenylalanine (Phe-2-O) and (S)-benzyltyrosine (Bz-Tyr-2-O) in pH gradients. With Bz-Tyr-2-O, calcium was generally favoured at every pH, the transport rate strongly depending upon the ligand concentration. At pH 8, a reversal of selectivity (Na+/Ca2+) was observed for the first time at low ligand concentration, thus suggesting that a supramolecular-type of transport occurs as a consequence of the different assemblages formed by the ligand according to pH and concentration. This is the first case of M2+/M+ selectivity induced by the carrier concentration.

Binding, Competitive↗

PubMed1989

pH regulation of calcium recognition by an amino acid containing acyclic ionophore.

Calcium ion recognition by a dicarboxylic ionophore containing two (S)-phenylalanine residues joined via an amide bond by a flexible tri-oxa-undecanoyl bridge (Phe-3-O) has been investigated in a wide pH range (from pH 2 to 12). Experiments were performed in methanol and chloroform by 1H and 13C NMR, relaxation times and 2D NMR (COSY, NOESY and J-resolved experiments). Recognition is shown to be regulated by pH, as it occurs in at least three different coordination modes according to the experimental conditions. Even at low pH (pH 2) the ion is already complexed in the compartment created by the ethereal oxygens and the amide carbonyls. At higher pH, it becomes fully encapsulated in a pseudo-cyclic structure and at very basic pH it is localized between the amide carbonyls and the carboxylates. For these peculiar properties Phe-3-O appears to be a very promising ionophore in a trans-membrane pH gradient system.

Calcium↗

PubMed1988

Copper(II) complexes of diamino-diamido-type ligands as chiral eluents in the enantiomeric separation of D,L-dansylamino acids by reversed-phase high-performance liquid chromatography.

For purpose of studying the mechanism of chiral recognition in high-performance liquid chromatography (HPLC) (reversed phase), a model system is proposed, i.e., copper(II) complexes of diamino-diamido-type ligands containing L-amino acids (AA-NN-n) which added to the eluent are able to perform the enantiomeric resolution of dansylamino acids on C8 and C18 columns. Being potentially tetradentate, this system should be more liable to give an apical or outer-sphere interaction with the enantiomer rather than a simultaneous dechelation of two binding sites, as in the classical ligand-exchange mechanism. This model allows the variation of several parameters, such as the structural and electronic features of the initial complexes, their relative stabilities and lipophilicities and their correlation with enantioselectivity in HPLC.

Amino Acids↗

PubMed1988

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.

Amino Acids↗