ESI-mass spectrometry analysis of unsubstituted and disubstituted β-cyclodextrins: fragmentation mode and identification of the AB, AC, AD regioisomers

AbstractThe study of unsubstituted and disubstituted β-cyclodextrins (β-CDs) by ESI-mass spectrometry is reported, applying a cone-induced fragmentation in the presence of a twofold excess of sodium chloride, in order to gain information about the fragmentation of the different regioisomers. On the basis of the fragmentation pattern observed for the unsusbstituted β-CD, a statistical model shows that the fragments generated by every regioisomer of a disubstituted CD (AB, AC, and AD) are expected to differ in their relative intensity and, therefore, they can be used for correctly identifying the three different regioisomers. The model was tested on the three regioisomeric (AB, AC, and AD) diamino-β-CDs and ditosyl-β-CD and on the AC and AD regioisomers of dimesitylenesulphonyl-β-CD, allowing in every case through statistical analysis of the fragmentation pattern the correct assignment of every regioisomer on the basis of an ESI mass spectrum (single quadrupole analyzer, high cone voltage) of the pure compounds. The absolute intensities of the fragmentation peaks were voltage-dependent but their ratios was voltage-independent, indicating that no mass bias in peak ratios is introduced by the analyzer. Given the fast time of analysis and its general applicability, independently from the substituents, we propose our method as an easy way to identify the regioisomers of disubstituted β-CDs

The Diverse Potential of Gluten from Different Durum Wheat Varieties in Triggering Celiac Disease: A Multilevel In Vitro, Ex Vivo and In Vivo Approach

The reasons behind the increasing prevalence of celiac disease (CD) worldwide are still not fully understood. This study adopted a multilevel approach (in vitro, ex vivo, in vivo) to assess the potential of gluten from different wheat varieties in triggering CD. Peptides triggering CD were identified and quantified in mixtures generated from simulated gastrointestinal digestion of wheat varieties (n = 82). Multivariate statistics enabled the discrimination of varieties generating low impact on CD (e.g., Saragolla) and high impact (e.g., Cappelli). Enrolled subjects (n = 46) were: 19 healthy subjects included in the control group; 27 celiac patients enrolled for the in vivo phase. Celiacs were divided into a gluten-free diet group (CD-GFD), and a GFD with Saragolla-based pasta group (CD-Sar). The diet was followed for 3 months. Data were compared between CD-Sar and CD-GFD before and after the experimental diet, demonstrating a limited ability of Saragolla to trigger immunity, although not comparable to a GFD. Ex vivo studies showed that Saragolla and Cappelli activated immune responses, although with great variability among patients. The diverse potential of durum wheat varieties in triggering CD immune response was demonstrated. Saragolla is not indicated for celiacs, yet it has a limited potential to trigger adverse immune response

Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model

Mesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. They possess intrinsic therapeutic potential and can also be in vitro manipulated and engineered to produce therapeutic molecules that can be delivered to the site of diseases, through their capacity to home pathological tissues. We have recently demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth

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 diami–nomethane dihydrochloride in dioxane in the presence of EtsN. 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

Reversed-phase liquid chromatography 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:CH3CN 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

Two-dimensional high-performance liquid chromatographic system for the determination of enantiomeric excess in complex amino acid mixtures

An HPLC system that allows the determination of the enantiomeric composition of complex mixtures of aminoacids such as those occurring in biological fluids (e.g., serum, cerebrospinal fluid) and foods is described. d- and l-aminoacids (including proline) can be determined. First, aminoacid 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 aminoacid can be switched to a reversed-phase column (C18) 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- aminoacids 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 aminoacids