Chitosan derivatives as dynamic coatings for transferrin glycoform separation in capillary electrophoresis.

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.

Hair testing applied to the assessment of in utero exposure to drugs: Critical analysis of 51 cases of the University Hospital of Verona.

The work discusses the results of hair and urine testing performed in 51 cases of suspected in utero drug exposure handled at the University Hospital of Verona from 2016 to 2022. On the day of birth or the day after birth, urine from mother and newborn (UM and UN) and hair from mother (HM), newborn (HN) and father (HF), if possible, were collected. Urine underwent immunoassay and GC-MS analysis, whereas hair underwent LC-MS/MS and GC-MS/MS analysis. In 50 out of 51 cases, HM and/or HN were available. In 92% of them, hair testing resulted in a positive, often (>50% cases) for more than one class of substance. The most detected substances were cocaine, opiates, methadone and cannabinoids. Maternal segmental analysis showed a prevalent decreasing concentration trend during pregnancy in case of positivity for one class of substances, whereas, as expected, a neatly prevalent increasing trend in the case of positivity for more than one class of substances. In nine cases, HF was also available, resulting in all being positive, usually for the same classes of substances identified in HM, thus questioning parental responsibility. In 33 cases, urine samples from the mother or newborn were also collected. Of them, 27 cases (82%) tested positive, showing peri-partum drug consumption and then confirming the severity of the addiction. Hair testing showed to be a reliable diagnostic tool to investigate in utero drug exposure because of the possibility of obtaining a complete picture of maternal addictive behaviour and family background, thanks to segmental maternal hair analysis and father hair testing.

Optimization and validation of a new approach based on CE-HRMS for the screening analysis of novel psychoactive substances (cathinones, phenethylamines, and tryptamines) in urine.

The continuous introduction in the market of new psychoactive drugs (NPS) represents a well-known international emergency. Indeed, the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime are paying great attention to the spread of NPS. In addition to the traditional analytical approaches based on GC-MS and HPLC-MS, also CE coupled with MS has proved to be a precious tool for the toxicological screening of biosamples. On these grounds, the aim of the present work was to test the application of CE-HRMS as a new screening tool for the rapid detection of these novel drugs in urine. Separations were performed in an uncoated fused-silica capillary with id of 75 µm with a total length of 100 cm, by applying a constant voltage of 15 kV. The QTOF-MS was implemented with an electrospray ion source operating in positive ionization full scan mode in the range of 100-1000 m/z. Under these conditions, different NPS has been tested, including eight cathinones, five phenethylamine, and seven tryptamines. The method was validated after optimization of the following analytical parameters: BGE composition and pH, separation voltage, sheath liquid composition, and flow rate and ESI source settings. The applicability of the method was successfully tested by analyzing a series of real urine samples obtained from drug users.

Asialo-transferrin: Biochemical aspects and association with alcohol abuse investigation.

Asialo-human transferrin (asialo-hTf) is a glycoform of the human serum protein transferrin characterized by the lack of the sialic acid (SA) terminal unit. It is known that glycosylation micro-heterogeneity and the presence of SA are strongly involved in protein functioning and pathophysiological activities. Some hTf glycoforms are valuable biomarkers for the detection of both genetic defects of glycosylation and/or sialoform distribution changes. The detection of the carbohydrate deficient transferrin (CDT) glycoforms is currently a widely employed method for the diagnosis of chronic alcohol abuse. The physiological significance of asialo-hTf is still unclear, despite its important biological implications. The current knowledge suggests that asialo-hTf may be involved in regulation of iron transport and release at the hepatic level, which, consequently, could strongly be affected by alcohol consumption. For these reasons, a deeper understanding of asialo-hTf structure and its physiological role is required, and an improved method of its analysis would favor the detection of both chronic abuse and other habits of alcohol intake and/or misuse. Thus, suitable analytical methods possessing higher sensitivity and specificity in comparison with the currently available techniques are certainly recommended. The present review summarizes the studies on asialo-hTf structure, roles, and detection techniques mainly in relation to its possible use as a potentially additional useful biomarker of alcohol abuse, and underlines its prospective value as a forensic and diagnostic tool.

Capillary Electrophoresis (CE) vs. HPLC in the determination of asialo-Tf, a crucial marker for the reliable interpretation of questioned CDT increases.

BACKGROUND AND AIM: CDT is a collective biomarker including asialo- and disialo-Tf, but researchers have generally focused attention on disialo-Tf, because of its easier detectability, since asialo-Tf is typically not detectable by the current methods in abstinent individuals, social drinkers and in many alcohol abusers with moderate CDT increases. In the search of a confirmation marker of alcohol-related CDT increases, the detectability of asialo-Tf was re-evaluated comparatively by using CE vs. HPLC. METHODS: 468 serum samples compulsorily drawn in a forensic/administrative context were analyzed by CE and HPLC to compare their sensitivity towards asialo-Tf. RESULTS: CE allowed the identification of asialo-Tf in 108 out of 165 CDT "positive" cases, based on disialo-Tf measurement (cut-off 1.8%). HPLC showed a detectable asialo-Tf peak only in 2 cases. In addition, in some cases of disputed CDT increases, the quasi-absence of this Tf component in front of an important increase of disialo-Tf allowed the ruling out of a diagnosis of alcohol abuse, in agreement with all other clinical and laboratory data. CONCLUSIONS: The present work shows a superior performance of CE vs. HPLC for the determination of asialo-Tf and the importance of this CDT component to avoid misinterpretation of non-alcohol related CDT increases.

A new sample treatment for asialo-Tf determination with capillary electrophoresis: an added value to the analysis of CDT.

BACKGROUND AND AIM: The non-glycosylated glycoform of transferrin (Tf), known as asialo-Tf, was not selected (in favor of disialo-Tf) as the measurand for the standardization of carbohydrate deficient transferrin (CDT) determination because of a lower diagnostic sensitivity provided with the currently available analytical procedures for sera. However, asialo-Tf could provide an additional value to disialo-Tf in the CDT analysis employed in forensic toxicology contexts. The present work aimed at developing an easy sample preparation based on PEG precipitation in order to improve the detectability of asialo-Tf in capillary electrophoresis (CE). METHODS: Equal volumes (35 µL) of serum and of 30% PEG-8000 were mixed and briefly vortexed. After centrifugation, the supernatant was iron saturated with a ferric solution (1:1, v/v). The mixture was analyzed in CE for asialo-Tf and disialo-Tf determination. RESULTS: PEG-8000 precipitation allowed the improvement of the baseline in the electropherograms in terms of interferences reduction particularly in the asialo-Tf migration region. The detection of asialo-Tf was possible in 89% of samples with disialo-Tf above the cut-off limit, whereas only 16% of them showed asialo-Tf by employing the traditional sample preteatment. CONCLUSIONS: Asialo-Tf represents an additional value to disialo-Tf as a biomarker of alcohol abuse in forensic toxicology.