Preliminary Study to Develop an Alternative Method for the Non-targeted Determination of Xenobiotics in Food by Means of Ultra High Performance Liquid Chromatography Coupled to High Resolution and Accuracy Mass Spectrometry

This preliminary study describes the use of high resolution and accuracy mass spectrometry techniques combined with new generation chemical software products for detecting and identifying contaminants in food commodities. As a first step, the extracts of routine target analysis samples (obtained in our official laboratory responsible for food residues control) were acquired and processed with this method in order to search unknown and non-targeted contaminants in food. In order to verify the feasibility of the presented method, the research has been firstly addressed to untargeted pesticides and their metabolites in stone fruits commodities and tomatoes. The differential analysis carried with Compound Discoverer 2.0 between the investigated unknown sample and the blank matrix sample allowed to remove all the matrix molecular components; Aggregated Computational Toxicology Resource (ACToR) helped to understand and predict chemical interpretation of substances. The acquisition in FullScan-AIF and FullScanddMS2 allowed the clear detection and identification of isobaric compounds such as quinalphos and phoxim. In order to verify that the proposed method is suitable to the scope of application, the main points of SANTE/11813/2017 Document have been followed. The results demonstrate that no false positives and no false negatives have been detected from the analysis of samples spiked with pesticides at 0.010 and 0.10 mg kg−1. This preliminary study has been also tested with a Proficiency Test (EUPT-FV-SM08) and, according to EUPT-FV-SM08 Final Report, our laboratory has been included in the 67% (56) that clearly detected over 70% pesticides. Finally, this method has been extended to other matrices and contaminants

Direct Conjugation of Resveratrol on Hydrophilic Gold Nanoparticles: Structural and Cytotoxic Studies for Biomedical Applications

Strongly hydrophilic gold nanoparticles (AuNPs), functionalized with citrate and L-cysteine, were synthetized and used as Resveratrol (RSV) vehicle to improve its bioavailability. Two different conjugation procedures were investigated: the first by adding RSV during AuNPs synthesis (1) and the second by adding RSV after AuNPs synthesis (2). The two different conjugated systems, namely AuNPs@RSV1 and AuNPs@RSV2 respectively, showed good loading efficiency (η%): η1 = 80 ± 5% for AuNPs@RSV1 and η2 = 20 ± 3% for AuNPs@RSV2. Both conjugated systems were investigated by means of Dynamic Light Scattering (DLS), confirming hydrophilic behavior and nanodimension (<2RH> 1 = 45 ± 12 nm and <2RH> 2 = 170 ± 30 nm). Fourier Transform Infrared Spectroscopy (FT-IR), Synchrotron Radiation induced X-Ray Photoelectron Spectroscopy (SR-XPS) and Near Edge X-ray Absorption Fine Structure (NEXAFS) techniques were applied to deeply understand the hooking mode of RSV on AuNPs surface in the two differently conjugated systems. Moreover, the biocompatibility of AuNPs and AuNPs@RSV1 was evaluated in the concentration range 1.0–45.5 µg/mL by assessing their effect on breast cancer cell vitality. The obtained data confirmed that, at the concentration used, AuNPs do not induce cell death, whereas AuNPs@RSV1 maintains the same anticancer effects as the unconjugated RSV

A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gramscale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment ofmonophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates

A Semisynthetic Approach to New Immunoadjuvant Candidates: Site-Selective Chemical Manipulation of Escherichia coli Monophosphoryl Lipid A

A semisynthetic approach to novel lipid A derivatives from Escherichia coli (E. coli) lipid A is reported. This methodology stands as an alternative to common approaches based exclusively on either total synthesis or extraction from bacterial sources. It relies upon the purification of the lipid A fraction from fed-batch fermentation of E. coli, followed by its structural modification through tailored, siteselective chemical reactions. In particular, modification of the lipid pattern and functionalization of the phosphate group as well as of the sole primary hydroxyl group were accomplished, highlighting the unusual reactivity of the molecule. Preliminary investigations of the immunostimulating activity of the new semisynthetic lipidA derivatives show that some of them stand out as promising, new immunoadjuvant candidates