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

Editorial: Advances in therapeutic drug monitoring of psychiatric subjects: Analytical strategies and clinical approaches

Despite the long and sometimes enthusing history of pharmacological therapy in psychiatry, the hard truth is that unfortunately a quite large percentage of patients is still not responding, or poorly responding, to treatment, leading to many life years lost to disability, many lost lives, and an immeasurable amount of suffering from patients, relatives, friends, and caregivers alike. Thus, any scientific advance and any practice that could lead to even a slight increase in psychiatric therapy effectiveness would also bring with them enormous benefits for both citizens and healthcare institutions. It is a strong conviction, after many years of practice and study, that therapeutic drug monitoring (TDM) is one of these practices, one that is continually advancing and progressing both from the analytical and clinical points of view, toward the final goal of better, personalized, precision medicine

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

Threat or treat: Exposure assessment and risk characterisation of chemical contaminants in soft drinks and chocolate bars in various Polish population age groups

In the frame of the European Food Risk Assessment (EU-FORA) fellowship programme, two studies on chemical contaminants in food matrices were carried out in Warsaw, Poland, at the Department of Food Safety and Chemical Analysis, Institute of Agricultural and Food Biotechnology. The first study addressed health concerns about the dietary exposure to bisphenol A (BPA) contamination due to consumption of soft drink by Polish population. BPA is an organic additive used in the production of epoxy resins and polycarbonate plastics and because of this it is used in the internal coating of cans and in plastic bottle production. Depending on several factors, BPA can migrate from these materials to the soft drink and so, it can be ingested by consumers causing hormonal and reproductive disorders. To estimate the Polish population exposure to BPA, several soft drinks belonging to different brands were purchased from a supermarket in the city of Warsaw and analysed. The result of the analysis highlight that mean BPA exposure in the Polish population exceeds the tolerable daily intake proposed by the EFSA scientific opinion, raising health concerns. On the other hand, the second study, focused on cadmium exposure due to chocolate consumption by Polish population, did not raise any health concern. Cadmium is a heavy metal that naturally occurs in its inorganic form in the environment and its presence in chocolate derives only from the cocoa beans and not from contamination during processing. Its accumulation in the human body can create several adverse effects, including renal dysfunction and failure. To estimate the Polish population exposure to cadmium, several chocolate bars were purchased from a supermarket in the city of Warsaw and analysed. The results of the analysis show that cadmium exposure in the Polish population does not exceed the tolerable weekly intake proposed by the EFSA scientific opinion

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

Evaluation of the Antioxidant Capacity of Fruit Juices by Two Original Analytical Methods

Two analytical methods previously developed by our groups were employed to estimate the antioxidant capacity of commercial fruit juices. The electrochemical method, which measures the scavenging activity of antioxidants towards OH radicals generated by both hydrogen peroxide photolysis and Fenton’s reaction, is based on the recovery of the cyclic voltametric response of the redox probe Ru(NH3)63+ at a Glassy Carbon electrode modified with a thin film of an insulating polyphenol, in the presence of compounds with antioxidant properties. The values of the antioxidant capacity of the fruit juices are expressed as vitamin C equivalents/L. The chromatographic method is based on the generation of OH radicals via Fenton’s reaction in order to test the inhibition of their formation in the presence of antioxidant compounds by monitoring salicylate aromatic hydroxylation derivatives as markers of •OH production, by means of HPLC coupled to coulometric detection. The results are expressed as the percentage of inhibition of •OH production in the presence of the tested juice compared to the control sample. When OH radicals are produced by Fenton’s reaction, the antioxidant capacity of the juices, estimated by both methods, displays an analogous trend, confirming that they can be considered an alternative for measuring the ability of antioxidants to block OH radical formation

Assessment of capillary volumetric blood microsampling for the analysis of central nervous system drugs and metabolites

Therapeutic drug monitoring (TDM) is an important tool for correlating the administered drug dose to drug and metabolite concentrations in the body and to therapeutic and adverse effects. In the case of treatment with drugs active on the central nervous system (CNS), frequent TDM becomes really useful, especially for patient compliance checking and for therapy optimisation. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine and sertraline, chosen as target compounds for this study, are two antidepressants mainly used for major depression, but also for obsessive-compulsive disorder associated with neurodegenerative diseases and for eating disorders. Microsampling approaches can be used to make TDM patient-friendly, by means of minimally invasive fingerpricking instead of classic invasive venipuncture. In this study, an innovative volumetric microsampling approach based on the use of hemaPEN technology is proposed to simultaneously obtain four identical dried whole blood microsamples by means of a single capillary sampling. The developed strategy shows significant advantages in terms of blood collection and storage, fast and feasible extraction procedure and sensitive LC-MS/MS analysis, also providing satisfactory validation results (extraction yield >81%, RSD <12.0%, and <6.3% loss in analyte stability after 3 months). The proposed methodology has proven to be sound and reliable for application to the TDM of psychiatric patients treated with antidepressant drugs such as fluoxetine and sertraline. The original capillary volumetric microsampling procedure using hemaPEN has been demonstrated to be suitable for the accurate sampling of capillary whole blood, in order to be successfully exploited in self- and home-sampling procedures in future and to pave the way for precision medicine approaches for the treatment of CNS disorders