Development and application of microsampling approaches.

The talk presented a comprehensive overview of the Volumetric Absorptive Microsampling (VAMS) approach, including the procedures followed and the results obtained by researchers who have worked on this analytical field in recent years. Various aspects were covered, such as sample collection, handling, and analysis. It may discuss the advantages and limitations of VAMS compared to other microsampling techniques, providing insights into the potential benefits and practical considerations for its implementation. The talk may also showcase specific studies or experiments conducted by different authors, highlighting their methodologies, experimental setups, and the outcomes they achieved. By presenting this information, the talk serves as a valuable resource for scientists seeking to explore and contribute to the field of microsampling.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Blood and Plasma Volumetric Absorptive Microsampling (VAMS) Coupled to LC-MS/MS for the Forensic Assessment of Cocaine Consumption

Reliable, feasible analytical methods are needed for forensic and anti-doping testing of cocaine and its most important metabolites, benzoylecgonine, ecgonine methyl ester, and cocaethylene (the active metabolite formed in the presence of ethanol). An innovative workflow is presented here, using minute amounts of dried blood or plasma obtained by volumetric absorptive microsampling (VAMS), followed by miniaturized pretreatment by dispersive pipette extraction (DPX) and LC-MS/MS analysis. After sampling 20 \ub5L of blood or plasma with a VAMS device, the sample was dried, extracted, and loaded onto a DPX tip. The DPX pretreatment lasted less than one minute and after elution with methanol the sample was directly injected into the LC-MS/MS system. The chromatographic analysis was carried out on a C8 column, using a mobile phase containing aqueous formic acid and acetonitrile. Good extraction yield (> 85%), precision (relative standard deviation, RSD < 6.0%) and matrix effect (< 12%) values were obtained. Analyte stability was outstanding (recovery > 85% after 2 months at room temperature). The method was successfully applied to real blood and plasma VAMS, with results in very good agreement with those of fluid samples. The method seems suitable for the monitoring of concomitant cocaine and ethanol use by means of plasma or blood VAMS testing

Hyphenated Approaches for the Analysis of Bioactive Natural Compounds in Complex Matrices

Plants, animals and micro-organisms represent a reservoir of natural products, the so called “natural source-derived compounds”. This is particularly true for the plant kingdom, as it offers a variety of species still used as remedies for several diseases in many parts of the world. Nevertheless, the bioactive potential of many plant species remains largely unexplored. Thus, biodiversity represents an unlimited source of chemical entities with potential beneficial effects on human health. These compounds are usually secondary metabolites often present in low quantity in plant material and their extraction, purification and quantitation still remain a great challenge for analytical scientists. The research activity carried out during these three years of PhD Programme was focused on the development, validation and application of original methods aimed at the quali-quantitative analysis of compounds with potential bioactive interest in plant matrices, foods, drinks and related products, as well as the analytical screening of plant by-products from cosmetic manufacture. Bioactive substances, belonging to the classes of polyphenols, aminoacids, coumarins, triterpenes and phytohormones, have been investigated as authenticity markers, in order to identify high quality products and to valorise niche products. The study regarded herbs (Argania spinosa), fruits (Citrus × myrtifolia, Punica granatum) and berries (Myrtus communis) mainly used as folk medicines for their broad spectrum of supposed pharmacological and therapeutic effects. The analytical methods developed within this study are based on high performance liquid chromatography and ultra-high performance liquid chromatography coupled to spectrofluorometric detection, triple quadrupole and high-resolution triple quadrupole mass spectrometry (HPLC-F, LC-MS/MS and UHPLC-HRMS). Significant efforts have been put also into the development and optimisation of miniaturised sample pretreatment strategies, such as micro-solid phase extraction (µSPE) and micro-extraction by packed sorbent (MEPS), able to purify complex matrices of natural origin (whole fruits, fruit parts, leaves and their extracts) and derived commercial products

Potential of hydrophobic paper-based sorptive phase prepared by in-situ thermal imidization for the extraction of methadone from oral fluid samples

Paper-based sorptive phases (PSPs) are functional planar materials with a demonstrated potential in analytical sample preparation. This article describes the synthesis of a polyimide coated paper by an in-situ imidization at a high temperature. Polyimides (PI) are synthesized in two subsequent steps where a hydrophilic polymer, in this case, poly(amic acid) (PAA), is formed as an intermediate product. PAA is finally transformed into hydrophobic PI by thermal curing at 180 °C. The synthesis of PI-paper takes advantage of this two-step procedure. In the first stage, a segment of filter paper is immersed into an aqueous PAA solution. After the solvent evaporation, the paper is heated at 180 °C for 1 h inducing the formation of the hydrophobic PI over the cellulose fibers. Infrared spectroscopy has been used to characterize the synthesized materials by defining a coverage factor F. The hydrophobicity of the materials has been studied using an aqueous methylene blue solution as a marker. To fully demonstrate the usefulness of the material in the sample preparation field, the extraction of methadone from oral fluid (OF) samples has been considered as a model analytical problem. The main variables affecting the synthesis (PAA concentration on the precursor solution and number of dips) and the extraction (elution and extraction times) have been fully evaluated. Working under the optimum conditions, a limit of quantification of 9 µg/L, intraday and interday precision better than 14.6%, and accuracy in the range of 87–108% were obtained

Green analytical chemistry (GAC) applications in sample preparation for the analysis of anthocyanins in products and by-products from plant sources

Agri-food industry manufacturing is an important source of environmental pollution and eutrophication, both intrinsically and due to the generation of significant amount of by-products. For this reason, green chemistry is currently at the forefront of efforts to make all steps of agri-food workflows more sustainable and environmentally friendly and to reduce their carbon footprint. Green analytical chemistry (GAC) is an integral part of these efforts, although it has been largely neglected until now, due to the fact that analytical procedures are mainly limited to quality control in this field, and thus produce just a small fraction of the overall environmental burden of agri-food processes. In this mini-review, the most recent developments of green analytical methods are described, relative to their applications for anthocyanin determination in agri-food products and by-products. Anthocyanins have been chosen as they are among the most valuable secondary plant metabolites, with a wide range of possible applications exploiting their preservative, antioxidant and coloring properties. Non-separative and separative analytical meth- ods are included in this mini-review. The former are mainly spectrometric in nature, and usually mostly allow to detect and/or quantify groups or classes of molecules. However, they also provide very high throughput and the greatest chance to develop low-energy, low-solvent consumption procedures, even to the point of enabling direct determinations in solid samples as such. On the other hand, separative methods provide far greater selectivity and far wider applicability, but at the price of higher energy and resource consumption and usually lower throughput

An Original HPLC Method with Coulometric Detection to Monitor Hydroxyl Radical Generation via Fenton Chemistry

Hydroxyl radicals (•OH) can be generated via Fenton chemistry catalyzed by transition metals. An in vitro Fenton system was developed to test both the inhibition and stimulation of •OH formation, by monitoring salicylate aromatic hydroxylation derivatives as markers of •OH production. The reaction was optimized with either iron or copper, and target analytes were determined by means of an original HPLC method coupled to coulometric detection. The method granted good sensitivity and precision, while method applicability was tested on antioxidant compounds with and without chelating properties in different substance to metal ratios. This analytical approach shows how Fenton's reaction can be monitored by HPLC coupled to coulometric detection, as a powerful tool for studying molecules' redox behavior

Enantioselective analysis of the methamphetamine precursors ephedrine and pseudoephedrine by capillary electrokinetic chromatography using cyclodextrins as chiral selectors

Ephedrine and pseudoephedrine can be used as precursors for the illicit synthesis of stimulant drugs, such as methamphetamine and methcathinone. For this reason, an enantioselective capillary electrokinetic chromatography method was developed for the separation of ephedrine and pseudoephedrine enantiomers. After testing two different charged or chargeable cyclodextrins (β-cyclodextrin sulphate and carboxymethyl-β-cyclodextrin) and a few mixtures as possible chiral selectors, a mixture of 3 mM carboxymethyl-β-cyclodextrin and 3 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin in pH 3.4 acetate buffer was used as the enantioselective background electrolyte and the separation was carried out in an uncoated silica capillary (75 µm internal diameter, 48.6 cm total length, 40.0 cm effective length). Analytical method performance was then evaluated in terms of linearity, precision, selectivity and accuracy with good results. Proof of concept application to mixtures simulating illicit methamphetamine (containing small amounts of ephedrine and pseudoephedrine as possible residual synthesis impurities) provided satisfactory results for the identification and quantitation of the four analyte diastereomers

Dried Volumetric Microsampling Approaches for the Therapeutic Drug Monitoring of Psychiatric Patients Undergoing Clozapine Treatment

Clozapine is one of the most widely used second-generation antipsychotic drugs (SGAs) for the treatment of schizophrenia. Despite advantages over first-generation drugs, clozapine still shows significant side effects and interindividual variations in efficacy. In order to ensure frequent therapeutic drug monitoring (TDM) and improve the compliance of psychiatric patients undergoing clozapine treatment, two novel dried microsampling approaches based on whole blood and plasma volumetric absorptive microsampling (b-VAMS and p-VAMS) and microfluidic generated-dried blood spot technology (mfDBS) were developed and coupled to HPLC with electrochemical detection (ED). The proposed miniaturized strategies by means of VAMS and microfluidic channel-based devices provide several advantages in terms of collection, storage, and handling compared to classical blood and plasma processing. Satisfactory validation results were obtained for all microsampling platforms, with mean extraction yields >85.1%, precision as relative standard deviation (RSD) < 5.1%, and stability < 4.5% analyte loss after 30 days for p-VAMS; mean extraction yields > 83.4%, precision RSD < 5.4%, and stability < 4.6% analyte loss after 30 days for b-VAMS, and mean extraction yields > 74.0%, precision RSD < 5.6%, and stability < 4.9% analyte loss after 30 days for mfDBS. The original microsampling methodologies have been successfully applied to the blood and plasma collected from five psychiatric patients for the monitoring of the levels of clozapine and its main metabolites, providing robust and reliable quali-quantitative results. Comparisons between results of the two dried microsampling technologies with those obtained by classic fluid plasma analysis were in good agreement and have demonstrated that the proposed miniaturized approaches could be suitable for TDM purposes

Microsampling and enantioselective liquid chromatography coupled to mass spectrometry for chiral bioanalysis of novel psychoactive substances

In this paper, the development of efficient enantioselective HPLC methods for the analysis of five benzofuran-substituted phenethylamines, two substituted tryptamines, and three substituted cathinones is described. For the first time, reversed-phase (eluents made up with acidic water-methanol solutions) and polar-ionic (eluent made up with an acetonitrile-methanol solution incorporating both an acidic and a basic additive) conditions fully compatible with mass spectrometry (MS) detectors were applied with a chiral stationary phase (CSP) incorporating the (+)-(18-crown-6)-tetracarboxylic acid chiral selector. Enantioresolution was achieved for nine compounds with α and RS factors up to 1.32 and 5.12, respectively. Circular dichroism (CD) detection, CD spectroscopy in stopped-flow mode and quantum mechanical (QM) calculations were successfully employed to investigate the absolute stereochemistry of mephedrone, methylone and butylone and allowed to establish a (R)<(S) enantiomeric elution order for these compounds on the chosen CSP. Whole blood miniaturized samples collected by means of volumetric absorptive microsampling (VAMS) technology and fortified with the target analytes were extracted following an optimized protocol and effectively analysed by means of an ultra-high performance liquid chromatography-MS system. By this way a proof-of-concept procedure was applied, demonstrating the suitability of the method for quali-quantitative enantioselective assessment of the selected psychoactive substances in advanced biological microsamples. VAMS microsamplers including a polypropylene handle topped with a small tip of a polymeric porous material were used and allowed to volumetrically collect small aliquots of whole blood (10 μL) independently from its density. Highly appreciable volumetric accuracy (bias, in the -8.7-8.1% range) and precision (% CV, in the 2.8-5.9% range) turned out

Epidemiological study in an area contaminated by chromium, arsenic, mercury and boron in Tuscany region

The Cecina river valley, in Tuscany, is characterized by environmental chromium contamination; Cr(VI) concentrations in ground and surface waters exceeds the WHO limit for drinking water. Boron, arsenic, mercury contaminations, of natural or industrial origin,pesticides use, and waste landfilling are causes of community concern. Statistically significant mortality excesses resulted for circulatory diseases in men(Obs=1788, SMR=1.06) and women (Obs=2111, SMR=1.10), colon cancer in women (Obs=44, SMR=1.38), stomach cancer in men (Obs=42, SMR=1.43). Statistically significant hospitalization excesses resulted for respiratory diseases in men (Obs=585, SHR=1.15), chronic respiratory diseases in women (Obs=88, SHR=1.71), digestive diseases in men (Obs=766, SHR=1.13) and women (Obs=565, SHR=1.13),lynphohematopoietic diseases in women (Obs=217, SHR=1.15). The results are useful to plan advanced environmental and epidemiological studies, to open dialogue with local representatives, to promote future remediation activities and to enforce recommendations