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

Haematic microsampling for monitoring psychiatric drugs: an overdose case study

In April 2018 some haematic samples, namely dried blood spots (DBS), plasma and serum were received at PTA Lab from the intensive care unit and the psychiatric diagnosis and care service of S. Orsola-Malpighi Hospital, coming from a psychiatric patient in a supposed state of intoxication for self-injurious purposes. It dealt with a complicated case of polypharmacy where quetiapine, an atypical antipsychotic used for the treatment of schizophrenia, bipolar disorder and major depressive disorders, was taken to attempt suicide in association with sertraline, a second-generation antidepressant belonging to the selective serotonin reuptake inhibitor (SSRI) category. An original HPLC method was applied within this non-fatal overdose case study to perform a comparative analysis on haematic samples processed by means of miniaturised approaches. Solid-phase microextraction fibers for liquid chromatography (LC-SPME) were used as a preliminary step to perform an analytical qualitative screening in a short time. Then, an advanced comparative analysis using microsamples was performed by means of dried matrix spot (DMS), featuring advantages over classic biological matrices in terms of simplified sampling, handling, and overall feasibility. Dried samples may be shipped and stored at room temperature instead of cryopreservation. Thus, the use of microsampling has multiple advantages in particular when limited volumes of biological samples are available. DBS, dried plasma spots (DPS) and dried serum spots (DSS) sampling and pretreatment were compared to a fully validated reference solid-phase extraction (SPE) on plasma and serum and proved to be less time-consuming, more straightforward and cost-effective, requiring lower matrix and solvent volumes. In fact, microsampling involved the use of 10 or 20 \ub5L of matrix deposited on specific cellulose supports; analyte extraction was performed by means of 500 \u3bcL of solvent exploiting a carefully optimised combination of fast microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). Within this bridging study, miniaturised dried matrices provided comparable findings with respect to the reference method, demonstrating the reliability of the quali-quantitative results obtained by microsampling. Moreover, they proved to be useful tools for application to the monitoring of patients in therapy with CNS drugs, especially when rapid screening and confirmation results are needed by using streamlined, feasible procedures yet providing reliable and sound data. The perspective of a fast analysis on a few blood drops directly collected from a fingertip places microsampling among the most promising approaches in bioanalysis, particularly relevant in the framework of clinical applications

An effective DBS-LC-MS/MS strategy for the monitoring of alcohol biomarkers

Workplace monitoring of alcohol consumption habits is highly advisable especially in the healthcare framework, due to the serious consequences alcohol use and abuse could bring. Within this project, two highly selective alcohol consumption biomarkers were selected, namely ethyl glucuronide (EtG) and ethyl sulfate (EtS), to perform a reliable quali-quantitative analysis as a tool to investigate alcohol consumption behaviours. The two analytes, as medium-term biomarkers, are suitable for workplace monitoring. To this aim, a minimally invasive biosampling strategy has been proposed and developed within this research, also allowing simplified sample storage and shipping of numerous samples: a dried blood spots (DBS) microsampling approach. It is based on haematic collection through a finger prick, avoiding an invasive procedure (i.e. phlebotomy). An accurate blood volume (10 \ub5L) was collected from a fingertip and spotted on special cellulose DBS cards; after spot drying, the cards were stored at room temperature without the need for cryopreservation. The use of a miniaturised, dried biological matrix also ensures enhanced EtG and EtS stability in blood, when compared to the same analytes in classic fluid blood samples. On the other hand, the reduced volumes and the low expected analyte levels required the development and validation of a highly sensitive and selective instrumental analytical method: the microsampling approach was coupled to an original LC-MS/MS method. In the first phase of the project, an in-depth optimisation of mass spectrometry parameters was carried out and led to the definition of electrospray ionization (ESI) polarity, acquisition mode, fragmentation parameters, selection of mass ion transitions leading to the highest selectivity and sensitivity. Chromatographic conditions were also optimised for the simultaneous determination of both analytes in a short time (3.5 minutes) and validated according to the main International Guidelines. In the second phase of the research, the developed method was applied to evaluate the analyte levels in DBS from volunteers, after alcohol controlled dosing and aiming at discriminating between abstinence, accidental alcohol intake, acute and chronic consumption. At the same time, an attempt was made to define EtG and EtS baseline levels, as well as effective cut-off reference values. The original high-throughput analytical methodology proposed herein combines the advantages of microsampling for sample collection, transportation and storage with the high sensitivity and selectivity of LC-MS/MS. It has granted the successful analysis of samples from healthcare professionals, recruited in the last phase of the study, for the monitoring of workplace alcohol consumption. This study was funded by the Italian Ministry of Health within the Finalized Research Project 2011-2012 (RF-2011-02352096) and performed at the Pharmaco-Toxicological Analysis Laboratory (PTA Lab) of the Department of Pharmacy and Biotechnology (FaBiT), Alma Mater Studiorum - University of Bologna

Good things come in small packages: microsampling and miniaturised sample pretreatment in clinical, forensic and anti-doping settings

Methodological and technological advances in the field of bioanalysis have opened up the potential to use smaller sample volumes (microsamples) of virtually any kind of fluid biological matrix. The main advantages of miniaturised techniques lie in the minimization of their impact in a greener chemistry perspective, logistic savings, reduction of sample handling and procedure streamlining. In the case of microsampling, these advantages are combined with the benefits of minimal invasiveness and of remote, on-field and home-sampling. Moreover, microsamples in dried form could grant improved sample stability even when stored at room temperature, leading to increased result reliability and methodological cost-effectiveness. To date, the research frameworks in which miniaturised techniques are drawing major attention are clinical practice, forensic and anti-doping settings. A comparative overview will be provided on the most recent and cutting-edge strategies developed and implemented in the PTA Lab of the University of Bologna. Dried microsampling techniques including dried matrix spots (DMS) and volumetric absorptive microsamplig (VAMS) have been extensively studied for their potential in the therapeutic drug monitoring (TDM) of central nervous system drugs. They have also been applied to haematic and urinary matrices to investigate drugs of abuse and doping agents, performing comparative bridging studies with traditional bioanalysis. Moreover, miniaturised sample pretreatment approaches have been designed and developed to enhance analysis throughput and procedure feasibility. Microextraction by packed sorbent (MEPS), solid phase microextraction fiber tips and probes (LC-SPME), and stop-and-go extraction (StAGE) have been coupled to original LC-MS/MS methods and their performances have been studied and compared. This study is carried out as part of a research project funded by the 2017 World Anti-Doping Agency (WADA) Research Grants and in particular within the section \u201cDetection of doping substances/methods: methodologies in analytical chemistry\u201d [Research project reference number 17A20LM]

Blood microsampling for untargeted lipidomics

Lipidomics has become a good bioanalytical tool for biomarker identification in a wide range of diseases. To this aim, whole blood could be promisingly exploited to obtain as much information as possible, despite the well-known intrinsic problems of this biological matrix, mainly related to sampling invasiveness, pre-analytical manipulation and processing, when compared to plasma and serum. To overcome the disadvantages of classic whole blood analysis, two microsampling approaches are proposed: Dried Blood Spot (DBS) and Volumetric Absorptive Microsampling (VAMS). Both techniques allow the collection of small amounts of matrix (20 µL) in a minimally invasive way, directly by fingerprick followed by drying and storage at room temperature. After the loss of water, often responsible for degradation reactions, dried microsamples ensure analyte stability. In addition, VAMS device can absorb a fixed and highly reproducible whole blood volume by means of a hydrophilic polymer tip, regardless of haematocrit value. Fast and feasible original pretreatment procedures have been developed and optimised by testing different pure and mixture solvents and extraction means. To evaluate the best performances for each lipid class and sampling mode, 15 benchmark lipids have been chosen, being representative for the most abundant lipid categories. Quali-quantitative results by means of an originally developed high-resolution UHPLC-MS/MS method were processed and compared by using a multivariate data analysis approach. This allowed to define the best extraction protocol for DBS and VAMS and, after comparison with fluid blood, to suggest VAMS strategy as a promising alternative procedure for blood sampling for untargeted lipidomics

Microfluidic-based volumetric sampling as a haematocrit-independent DBS collection strategy

Central nervous system (CNS) drugs require accurate therapeutic drug monitoring (TDM) in a combined bioanalytical and clinical approach. TDM is performed in order to evaluate the suitability of prescribed therapies by measuring drug plasma concentrations to optimise dose regimen, minimise toxicity and for overall clinical decision-making. For this purpose, the use of microsampling, namely dried blood spots (DBS), involves considerable advantages compared to the classic in-tube plasma analysis approach, both for clinicians and patients: in fact, a minimally invasive sample collection allows more frequent and accurate monitoring. These advantages are in addition to those related to simplified handling and storage, fast processing and feasible analysis procedures. On the other hand, quali-quantitative results obtained by the DBS approach can be affected by volumetric bias due to blood haematocrit (HCT) and density, reflecting on spot size and homogeneity, sampling reproducibility, accuracy and precision of analytical data. For this reason, in-depth studies have been carried out in order to investigate the correlations between HCT values and DBS analysis results. To overcome HCT-dependent bias, an alternative strategy based on microfluidic devices has been proposed: a fixed, small volume (5-10 \ub5L) of whole blood is accurately collected in a microfluidic channel-based device to generate DBS samples regardless of blood viscosity. As a proof of concept, this innovative approach was designed and developed on aripiprazole, an atypical antipsychotic used in the treatment of schizophrenia and bipolar disorder. To this aim, an original analytical methodology was developed, validated and successfully applied for the analysis of DBS samples obtained by microfluidic devices from patients undergoing aripiprazole treatment. The resulting data has shown the novel microfluidic-based microsampling procedure grants accurate and precise results, irrespective of HCT. This research work provides an effective strategy based on microcapillaries to collect a fixed small volume of whole blood, which could overcome HCT biases in DBS and promises to be feasible and reliable while maintaining all the advantages of classic DBS analysis, making it suitable for widespread bioanalytical applications

Characterisation of bioactive compounds in wine by-products: toward the valorisation of sustainable resources

Grape vine (Vitis vinifera L.) is one of the most cultivated plants in the world. Regular consumption of grape products is associated with a reduction in the onset of chronic-degenerative diseases. In the European Union most of the harvest is used by the wine industry, generating a large amount of grape processing by-products. Pomace, seeds and stalks are the main processing residues obtained during the winemaking process and represent matrices that could be exploited for sustainable recycling. Aim of this multidisciplinary research project is the extraction, identification and quantitation of bioactive compounds in grape processing by-products, the evaluation of the biological effects of bioactive-enriched extracts and the mapping of the identified bioactive substances by comparing the profiles of different plant parts (pomace, seeds and stalks) and grape cultivars (Albana and Sangiovese). In order to obtain bioactive-enriched samples exploiting eco-sustainable technologies, extraction procedures were developed by means of solid/liquid-, ultrasound- and microwave-assisted extraction (SLE, UAE, MAE). An original LC-MS/MS methodology has been set up, fully validated and applied to the extracts in order to obtain a detailed quali-quantitative profiling of several compound classes (flavonoids, phenolic acids, procyanidins, stilbenoids). Moreover, all the extracts were evaluated for their therapeutic potential on gastrointestinal diseases by exploiting ex-vivo animal tissues, and then correlated to their quali-quantitative composition by computational approaches based on molecular networks. The comparative characterisation of bioactive compounds present in grape by-products could be the basis for the valorisation and integrated exploitation of this cheap and easily available alternative source in the nutraceutical, cosmeceutical and pharmaceutical fields. This research was funded by \u201cFondazione Cassa di Risparmio di Imola\u201d (Project code: 065.0318-2018.0050; Call year: 2017)

OEMONOM: Open access Educational Materials On Naturally Occurring Molecules - sources, biological activity and use

Background: Natural compounds have always attracted interest due to their potential effects on human being. Their use is widespread and is rather rising due to false belief that they are always better and safer than conventional drugs. The prepared materials should hence reportg both positive and negative aspects of natural compounds used for several commonly used indications (infections, menopause and gynaecological problems, benign prostatic hyperplasia, cough, cardiovascular diseases, CNS, skin and GIT disorders), as well as of vitamins and sympathomimetics, according to the most recent scientific evidence. Special materials will be also prepared for their interactions with conventional drugs. Summary: The Open access Educational Materials on Naturally Occurring Molecules \u2013 sources, biological activity and use (OEMONOM) project targets at preparation of comprehensible, free and easily available materials for professionals, students of biomedicinal disciplines as well as lay persons in relation to the effect of natural compounds on human health. The project arose from collaboration of 8 European universities and will be available in 8 native languages of participating universities (Czech, French, German, Hungarian, Italian, Portuguese, Slovak and Slovene). Materials will be prepared by common work of experts from different fields (pharmacologists, pharmacognostics, toxicologists, microbiologists, analytical chemists and E-learning experts)

Miniaturised alternative sampling for the TDM of eating disorder patients under antidepressant treatment

Eating Disorders (ED) represent a wide-ranging and growing phenomenon affecting the physical and psychic sphere of patients. In order to relieve anxiety and depression and reduce binge-eating behaviours associated with ED, treatments with antidepressant drugs (AD) are often needed. Therapies involving central nervous system (CNS) drugs require frequent and accurate therapeutic drug monitoring (TDM) to establish optimal individual prescribed dose, especially for patients with undernutrition or obesity, with physical comorbidities, suspected non-compliance or subject to adverse effects. To this aim, a microsampling approach with promising advantages over classic in-tube techniques has been developed and applied for this study. In fact, microsamples can be obtained in a minimally invasive way with the use of a few drops of capillary blood from a fingerprick. Moreover, after drying at room temperature, the samples do not require any cryopreservation for transport and storage. Dried microsamples usually show comparable stability profiles with respect to biological fluids stored at controlled temperature, due to the elimination of water as a substrate for enzymatic and bacterial degradation. Volumetric Absorptive Microsampling, a recent and promising development of miniaturised sampling, allows also to collect an accurate and reproducible micro-volume of biological fluid regardless of its density, by means of a polymeric porous tip allowing direct sampling from a fingerprick. Within this research project, two biological matrices were studied: whole blood as a classic biological matrix and oral fluid as an alternative one. Several AD drugs such as sertraline, fluoxetine, vortioxetine and their main active metabolites were investigated in both matrices, exploiting HPLC-UV/F/MS. It was then possible to compare the quali-quantitative results obtained from both the matrices and investigate the correlation between drug dosage, blood and oral fluid concentrations. All the original analytical procedures of miniaturised sampling, pretreatment and analysis have been developed and fully validated in order to achieve straightforward and high-throughput methodologies, to guarantee high reliability of the obtained data and the actual suitability of oral fluid and dried samples as alternative matrices for research and clinical practices