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Chitosan and its derivatives are interesting biopolymers for different field of analytical chemistry, especially in separation techniques. The present study was aimed at testing chitosan water soluble derivatives as dynamic coating agents for application to capillary electrophoresis. In particular, chitosan was modified following three different chemical reactions (nucleophilic substitution, reductive amination, and condensation) to introduce differences in charge and steric hindrance, and to assess the effect of these physico-chemical properties in capillary electrophoresis. The effects were tested on the capillary electrophoretic separation of the glycoforms of human transferrin, an important iron-transporting serum protein, one of which, namely disialo-transferrin (CDT), is a biomarker of alcohol abuse. Chitosan derivatives were characterized by using NMR and 1H NMR, HP-SEC-TDA, DLS, and rheology. The use of these compounds as dynamic coatings in the electrolyte running buffer in capillary electrophoresis was tested assessing the peak resolution of the main glycoforms of human transferrin and particularly of disialo-transferrin. The results showed distinct changes of the peak resolution produced by the different derivatives. The best results in terms of peak resolution were achieved using polyethylene glycol (PEG)-modified chitosan, which, in comparison to a reference analytical approach, provided an almost baseline resolution of disialo-transferrin from the adjacent peaks.

Chitosan films have attracted increased attention in the field of sensors because of chitosan’s unique chemico-physical properties, including high adsorption capacity, filmability and transparency. A chitosan film sensor was developed through the dispersion of an ammonia specific reagent (Nessler’s reagent) into a chitosan film matrix. The chitosan film sensor was characterized to assess the film’s properties by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A gas diffusion device was prepared with the chitosan film sensor, enabling the collection and detection of ammonia vapor from biological samples. The chitosan film sensor color change was correlated with the ammonia concentration in samples of human serum and artificial urine. This method enabled facile ammonia detection and concentration measurement, making the sensor useful not only in clinical laboratories, but also for point-of-care devices and wherever there is limited access to modern laboratory facilities.

Protein analysis by capillary electrophoresis is an established benchmark technique, where electrophoretic separation performances are frequently affected by protein adsorption on capillary walls. The protein-wall interaction can be suppressed or mitigated by the application of the so-called dynamic coating agents. In this study, the potential of chitosan derivatives as dynamic coating was investigated, targeting the separation of the carbohydrate deficient transferrin (CDT), a relevant biomarker in forensic analyses to detect chronic alcohol abuse. To this aim, chitosan was derivatized through different chemical reactions, including nucleophilic substitution, reductive amination, and condensation reaction, to provide different characteristics to the dynamic coatings. After chemical and physical characterization, chitosan derivatives were tested as dynamic coating additives in the buffer separation solution of CDT. Evidence of the improvement in the CDT separation efficiency were found applying PEGmodified chitosan via nucleophilic substitution.

This document describes the guidelines developed by the Scientific Association Gruppo Tossicologi Forensi Italiani (GTFI) for the determination of psychoactive substances in biological samples for forensic toxicology and medico-legal purposes. The guidelines provide indications and recommendations for performing forensic toxicological and medico-legal analyses with a high level of quality, with particular reference to the use of appropriate analytical techniques and established analytical procedures and methods shared by the national scientific community, in order to ensure results of high reliability and reproducibility. The correct methods for the determination of alcohol and psychoactive drugs in biological samples may also be of interest to other scientific communities, given the wide range of applications of the various toxicological examinations required and carried out not only for forensic purposes, but also for clinical-diagnostic purposes, the results of which may constitute evidence in administrative or criminal contexts. In this sense, and in the framework of an already existing collaboration between the GTFI and the Italian Society of Clinical Pathology and Laboratory Medicine (SIPMeL), the publication of an extract from the GTFI guidelines may also be useful for professionals belonging to these scientific societies who perform laboratory tests for various toxicological purposes. This document represents an abbreviated version of the original guidelines (Revision No. 6 of 2022), which can be accessed in full at the link https://www.gtfi.it/wp-content/uploads/2023/02/LineeGuidaGTFI-MaterialeBiologico-rev06-08giu2022.pdf, in order to keep the length within appropriate limits for a scientific journal. The sections most relevant to clinical laboratory professionals have been retained: 1. Purpose; 5. Requirements for Analytical Activities; 6. Acceptance, collection, handling and manipulation of specimens; 7. Analytical methods; 8.Analytical report.

The tear film is a complex matrix composed of several molecular classes, from small metal ions to macromolecules. Contact lens (CL) wear can affect the protein homeostasis of the tear film, by accumulating deposits on the CL surface and/or altering their structural and functional properties. This work investigates the effect of CL wear on lactoferrin (Lf), one of the most abundant tear proteins, known as an unspecific biomarker of inflammation. Tears from eight volunteers were collected and analyzed after alternated periods of CL wear and without CL. The experimental approach is to probe Lf into unprocessed human tears by the peculiar fluorescence emission originating from complex formation of Lf with terbium (Tb3+) at the iron-binding sites. The experimental data indicate that CL wear does not significantly affect the total amount of Lf. On the other hand, Lf affinity for Tb3+ is reduced upon CL wear, suggesting relevant changes in Lf structure and possible alterations of protein functionality. Future studies based on this approach will help define CL features (material, lens-care solution, wearing time, etc.) with minimal effects on tear protein activity, in order to obtain more biocompatible and comfortable devices.

Transferrin (Tf) is the major iron-transporting protein in the human body and, for this reason, has been extensively studied in biomedicine. This protein undergoes a complex glycosylation process leading to several glycoforms, some of which are important in the diagnosis of alcohol abuse and of congenital glycosylation defects under the collective name of carbohydrate-deficient transferrin (CDT). Exploiting the Tf ability to bind not only iron but also other ions, specific attention has been devoted to binding activity towards Tb3+, which was reported to greatly enhance its intrinsic fluorescence upon the interaction with Tf. However, the structural properties of the Tb3+-Tf complex have not been described so far. In the present work, the formation of the Tf-Tb3+complex has been investigated by the employment of several biophysical techniques, such as fluorescence resonance energy transfer (FRET), “native” mass spectrometry (MS), and near-UV circular dichroism (CD). Each method allowed the detection of the Tf-Tb3+complex, yielding a specific signature. The interaction of Tb3+with Fe3+-free Tf (apoTf) has been described in terms of stoichiometry, affinity, and structural effects in comparison with Fe3+. These experiments led to the first direct detection of the Tf-Tb3+complex by MS, indicating a 1:2 stoichiometry and allowing the investigation of structural effects of metal binding. Either Tb3+or Fe3+binding affected protein conformation, inducing structural compaction to a similar extent. Nevertheless, near-UV CD and pH-dependence profiles suggested subtle differences in the coordination of the two metals by Tf side chains. Experimental conditions that promote complex formation have been identified, highlighting the importance of alkaline pH and synergistic ions, such as carbonate. On the basis of these studies, sample pretreatment, separation, and detection conditions of a high-performance liquid chromatographic method for CDT analysis are optimized, achieving

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