Advances, challenges and future perspectives in microsampling-based bioanalysis

There is a growing interest in the design, development and implementation of miniaturised approaches for the determination of prescription drugs, drugs of abuse, doping agents, metabolites and biomarkers in biological samples involved in a wide range of applications and frameworks. This interest is due to the ethical advantages of sampling minute amounts of biological matrices, particularly for those studies performed in delicate populations and in the framework of patient-centric approaches. Moreover, these advanced technologies facilitate sampling to be performed in locations usually difficult to be reached, and allow for feasible, straight-forward and time- and cost-effective analytical protocols. This lecture gives a comprehensive overview about optimisation and implementation processes of cutting-edge sampling, pretreatment and analysis strategies in bioanalytical method development and application. In particular, the research group of Pharmaco-Toxicological Analysis (PTA Lab) of Alma Mater Studiorum - University of Bologna (Italy) recently designed and developed a panel of novel, miniaturised protocols to be applied for the determination of central nervous system drugs, drugs of and doping agents. The microsampling approaches fully designed and developed by PTA Lab include capillary volumetric blood microsampling, volumetric absorptive technologies and microfluidic platforms. These allow not only to collect microvolumes of biological matrices (including hematic samples, urine and oral fluid) in an accurate manner regardless of fluid density, but also to guarantee sample integrity, subject compliance and feasible, yet effective, pre-analytical and analytical steps. A comparative evaluation of procedures and techniques offering peculiarities, advantages and challenges will be presented and could guide attendees towards the best miniaturisation choice in relation to the different bioanalytical application scenarios

Microsampling-based analysis: principles and technologies.

Volumetric absorptive microsampling (VAMS) is an innovative technique that has gained attention in the field of bioanalysis. It allows the collection of blood and other biological samples for various analytical purposes. While VAMS shares similarities with the well-established dried blood spot (DBS) technique, it offers several advantages that make it a promising alternative. One of the key advantages of VAMS is improved accuracy in sampling volume. With DBS, the volume of the collected blood sample can vary depending on factors such as the hematocrit (HCT) level, leading to potential inaccuracies in analysis. VAMS, on the other hand, overcomes this limitation by enabling consistent and precise volumetric sampling, regardless of the HCT level. This ensures more reliable and reproducible results. Additionally, VAMS reduces the need for pre-treatment of samples. In the DBS technique, the blood spots often require additional steps such as punching, extraction, and elution to extract the analytes of interest. VAMS simplifies the process by allowing direct analysis of the absorbed sample, eliminating or minimizing the need for complex pre-treatment steps. This saves time, reduces the risk of sample contamination, and improves overall efficiency. Although VAMS shows promise, there are still aspects that require further investigation and optimization. Researchers and scientists are actively exploring its full potential, addressing challenges and refining protocols to ensure its reliability and applicability across various analytical platforms.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

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

Berberine and Tinospora cordifolia exert a potential anticancer effect on colon cancer cells by acting on specific pathways

Berberine (BBR) is a natural active principle with potential antitumor activity. The compound targets multiple cell signaling pathways, including proliferation, differentiation, and epithelial-mesenchymal transition. The aim of this study was to elucidate the mechanisms behind the anticancer activity of BBR by comparing the effects of purified BBR with those of the extract of Tinospora cordifolia, a medicinal plant that produces this metabolite. The expression levels of a panel of 44 selected genes in human colon adenocarcinoma (HCA-7) cell line were quantified by real-time polymerase chain reaction (PCR). BBR treatment resulted in a time- and dose-dependent down regulation of 33 genes differently involved in cell cycle, differentiation, and epithelial-mesenchymal transition. The trend was confirmed across the two types of treatment, the two time points, and the different absolute dosage of BBR. These findings suggest that the presence of BBR in T. cordifolia extract significantly contributes to its antiproliferative activity