Reduced Sample Volume Research Articles

Portable Fluorescence Microarray Reader-Enabled Biomarker Panel Detection System for Point-of-Care Diagnosis of Lupus Nephritis

Point-of-care (POC) testing has revolutionized diagnostics by providing rapid, accessible solutions outside traditional laboratory settings. However, many POC systems lack the sensitivity or multiplexing capability required for complex diseases. This study introduces an LED-based fluorescence reader designed for POC applications, enabling multiplex detection of lupus nephritis (LN) biomarkers using a biomarker microarray (BMA) slide. The reader integrates an LED excitation source, neutral density (ND) filters for precise intensity control, and onboard image processing with Gaussian smoothing and centroid thresholding to enhance signal detection and localization. Five LN biomarkers (VSIG4, OPN, VCAM1, ALCAM, and TNFRSF1B) were assessed, and performance was validated against a Genepix laser-based scanner. The LED reader demonstrated strong correlation coefficients (r = 0.96–0.98) with the Genepix system for both standard curves and patient samples, achieving robust signal-to-noise ratios and reproducibility across all biomarkers. The multiplex format reduced sample volume and allowed simultaneous analysis of multiple biomarkers. These results highlight the reader’s potential to bridge the gap between laboratory-grade precision and POC accessibility. By combining portability, cost-effectiveness, and high analytical performance, this fluorescence reader provides a practical solution for POC diagnostics, particularly in resource-limited settings, improving the feasibility of routine monitoring and early intervention for diseases requiring comprehensive biomarker analysis.

A Droplet Microfluidic Sensor for Point-of-Care Measurement of Plasma/Serum Total Free Thiol Concentrations.

Total free thiols are an important marker of the whole-body redox state, which has been shown to be associated with clinical outcome in health and disease. Recent investigations have suggested that increased insight may be gained by monitoring alterations of redox state in response to exercise and hypoxia and to monitor redox trajectories in disease settings. However, conducting such studies is challenging due to the requirement for repeated venous blood sampling and intensive lab work. Droplet microfluidic sensors offer an alternative platform for developing a point-of-care testing approach using small sample volumes and automated systems to complement or ultimately replace laboratory testing. Here we developed a small, portable droplet microfluidic sensor that can measure total free thiol concentrations in 20 μL human plasma (or serum) samples, providing a reading in less than 10 min. This system features a novel method to enhance the mixing of reagent and analyte in droplets containing viscous biological fluids. The results in a range of real-world human plasma samples showed equivalence with current standard laboratory assays while reducing sample volume requirements 9-fold and fully automating the process. Micro hematocrit capillaries allowed testing of capillary blood samples collected by fingerprick lancing. The system was used to monitor total free thiols using fingerprick samples in healthy volunteers and revealed significant changes in total free thiols in response to food intake and exercise. This device has the potential to improve our ability to conduct physiological studies of total free thiol level changes and improve our understanding of redox physiology, which may ultimately be applied in redox medicine to improve patient care.

Radiocarbon measurements of dissolved inorganic carbon (DIC) in sediment porewater and seawater at AWI MICADAS

Abstract Radiocarbon (14C) measurements on dissolved inorganic carbon (DIC) are a powerful tool to trace water masses and carbon cycling in the ocean. Existing methodologies to determine the 14C content of seawater DIC requires large volumes of sample (usually >100 mL) and specialized graphitization techniques to achieve the accuracy and precision needed for meaningful data interpretation. The advancement of the CO2 gas ionization accelerator mass spectrometry (AMS) technique today allows routine 14C measurements on small samples (<100 µgC) and may thus permit reducing the sample volumes needed to determine 14C content of seawater DIC to ∼2 mL. The proposed method utilizes the carbonate handling system (CHS), gas interface system (GIS) and MICADAS AMS, and provides good accuracy but reduced precision compared to established methods. Good accuracy is shown by comparing results for a marine in-house DIC standard and a DIC seawater profile from Antarctica between the proposed CHS-GIS-MICADAS approach and reference measurements conducted on the same material at established laboratories (ETH and NOSAMS). Further, two sedimentary porewater profiles from a fjord system in Svalbard are presented. Despite good agreement, the precision of the CHS-GIS-MICADAS approach is reduced, potentially limiting possible interpretations on seawater DIC. Nonetheless, the reduction of sample volumes proves particularly helpful to analyze porewater DIC from sediment cores, where sample material is notoriously limited, reduces the required amounts of toxic HgCl2 and simplifies expedition logistics.

Carboxylated Graphene: An Innovative Approach to Enhanced IgA-SARS-CoV-2 Electrochemical Biosensing.

Biosensors harness biological materials as receptors linked to transducers, enabling the capture and transformation of primary biorecognition signals into measurable outputs. This study presents a novel carboxylation method for synthesizing carboxylated graphene (CG) under acidic conditions, enhancing biosensing capabilities. The characterization of the CG was performed using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), Raman spectroscopy, thermogravimetric analysis (TGA), and X-ray diffraction (XRD). We modified screen-printed carbon electrodes (SPCEs) with CG to immobilize the SARS-CoV-2 N-protein, facilitating targeted detection of IgA antibodies (IgA-SARS-CoV-2). The analytical performance was assessed via electrochemical techniques such as cyclic voltammetry and electrochemical impedance spectroscopy, confirming CG synthesis effectiveness and biosensor functionality. The developed biosensor efficiently detects IgA-SARS-CoV-2 across a dilution range of 1:1000 to 1:200 v/v in a phosphate-buffered saline (PBS) solution, with a limit of detection calculated at 1:1601 v/v. This device shows considerable potential because of its fast response time, miniaturized design facilitated by SPCEs, reduced sample volume requirements, high sensitivity and specificity, low detection limits, and signal enhancement achieved through nanomaterial integration.

Evaluation of metabolite stability in dried blood spot stored at different temperatures and times

Dried blood spot (DBS) sampling offers significant advantages over conventional blood collection methods, such as reduced sample volume, minimal invasiveness, suitability for home-based sampling, and ease of transport. However, understanding the effects of variable storage temperatures and times on metabolite stability is crucial due to varying intervals and delivery conditions between sample collection and metabolomics analysis. To minimize biological variances, all samples were collected from the same individual simultaneously and stored at three different temperatures (4 °C, 25 °C, and 40 °C) for diverse time points (3, 7, 14, and 21 days). Metabolic profiling was conducted an untargeted gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)-based multi-platform metabolomics. Principal component analysis (PCA) showed alterations in metabolite stability at different temperatures, with phosphatidylcholines (PCs) and triglycerides (TAGs) as the first principal component (PC1). Specifically, we identified 69 metabolites that remained stable across all three temperatures over the 21-day period, while 78 metabolites exhibited instability. Furthermore, linear correlations between metabolite intensity and storage time were observed. Overall, our study elucidated the influence of storage temperature and time on specific metabolite stability in DBS samples, providing valuable insights for study design, biomarker selection, and data improvement.

NSE protein detection in a microfluidic channel integrated an electrochemical biosensor

This study proposed a microfluidic chip for the detection and quantification of NSE proteins, aimed at developing a rapid point-of-care testing system for early lung cancer diagnosis. The proposed chip structure integrated an electrochemical biosensor within a straight PDMS microchannel, enabling a significant reduction in sample volume. Additionally, a method was developed to deposit silver and silver chloride layers onto the reference electrode. Following fabrication, the working electrode was modified to immobilize NSE antibodies on its surface, facilitating specific protein detection. Electrochemical impedance spectroscopy (EIS) measurements were utilized to investigate the alterations in surface impedance resulting from the specific binding of anti-NSE on the electrode surface across varying concentrations of NSE, ranging from 10 ng ml-1to 1000 ng ml-1. The experimental results demonstrated a direct correlation between NSE concentration and surface impedance. Specifically, the charge transfer resistance exhibited an increase from 24.54 MΩ to 89.18 MΩ as the NSE concentration varied from 10 ng ml-1to 1000 ng ml-1. Moreover, the concentration of NSE can be quantified by relating it to the charge transfer resistance, which follows a logarithmic equation. The limit of detection (LoD) of the chip was evaluated to be approximately 1.005 ng ml-1. The proposed chip lays a crucial foundation for developing a Lab-on-a-chip platform dedicated to diagnosing NSE testing and lung cancer.

Development of an automated microfluidic system for actinide separation and analysis.

Mass spectroscopy and microfluidic technology, when combined, offer significant advantages in radiochemical analysis sample volume and cost reduction. A microfluidic device designed for efficiency has been developed. This device separates uranium from key trace elements by utilising UTEVA® chromatographic resins and nitric acid solutions of different concentrations for adsorption and recovery. The eluates from this microdevice are then diluted and directed to an inductively coupled plasma mass spectrometry system, enabling direct analysis of trace elements and uranium with minimal operator-sample interaction. This efficient approach greatly reduces the volume of sample required for trace elemental analysis in actinide materials, thereby reducing costs and satisfying the As Low As Reasonably Achievable (ALARA) principle.

Electrochemical Sensing for HIV Integrase Activity Detection Employing G4/Hemin Dnazyme

Introduction; HIV (human immunodeficiency virus) remains a major global public health issue, having claimed 40.4 million [32.9–51.3 million] lives so far with ongoing transmission in all countries globally. The World Health Organization (WHO) recommends a combination prevention approach [1], which uses a mix of biomedical, behavioral, and structural interventions, to have the greatest sustained impact on reducing new infections. Among the various strategies includes pre-exposure prophylaxis (PrEP), where people at risk of contracting HIV take antiretroviral medication in order to reduce the risk of HIV acquisition. To be effective, PrEP requires maintaining high adherence to antiretroviral medications, represented by HIV integrase inhibitors (INSTIs) [2]. Therefore, knowing an individual’s blood INSTI concentration is important to improve their adherence and to adjust individual dosage/administration methods accordingly for effective treatment. Currently, the HIV-1 Integrase assay kit is available, which is based on the monitoring of HIV-integrase DNA strand elongation activity. Using this assay, blood concentration of HIV-integrase inhibitors can be substantially monitored. However, the current procedures are complicated and require specific instruments, which make it difficult to apply for point-of-care testing and personal use. Accordingly, there is a growing interest in exploring alternative analytical methods to detect HIV-integrase activity that offer advantages such as reduced sample volume requirements, simplified procedures, and portability. In this presentation, we report the development of an electrochemical sensor for HIV integrase activity detection employing G4/hemin DNAzyme, focusing on the unique enzymatic reaction of HIV integrase, which will be utilized for the detection of inhibitors as well as the screening of novel inhibitors. Methods; The sensing principle of this sensor is based on the unique properties of HIV integrase, which recognizes the terminal regions of HIV genomic DNA and can integrase a dsDNA derived from HIV genomic DNA (Donor DNA) with any dsDNA (target DNA) [3]. To detect this activity, we designed target DNA sequence harboring DNAzyme sequence in the 5’ end, which shows peroxidase activity [4]. Accordingly, the amount of target DNA integrated into donor DNA can be monitored by peroxidase activity derived from DNAzyme, which is the subject of the electrochemical monitoring. Results and Discussion; Firstly, we evaluated the function of hemin aptamer-fused target DNA as a substrate of HIV integrase using fluorescence dye labeled hemin aptamer-fused target DNA. As a result, the fluorescence intensity increased depending on HIV integrase, so hemin aptamer-fused target DNA works as a substrate of HIV integrase. Secondly, we evaluated the peroxidase activity of the complex of hemin aptamer-fused target DNA. As a result, the complex retained peroxidase activity, indicating that the hemin aptamer-fused target DNA/hemin works as DNAzyme. The electrochemical detection of HIV integrase activity based on hemin aptamer-fused target DNA/hemin by chronoamperometry will be presented.

Resolving Structures of Paramagnetic Systems in Chemistry and Materials Science by Solid‐State NMR: The Revolving Power of Ultra‐Fast MAS

AbstractUltra‐fast magic‐angle spinning (100+kHz) has revolutionized solid‐state NMR of biomolecular systems but has so far failed to gain ground for the analysis of paramagnetic organic and inorganic powders, despite the potential rewards from substantially improved spectral resolution. The principal blockages are that the smaller fast‐spinning rotors present significant barriers for sample preparation, particularly for air/moisture‐sensitive systems, and are associated with low sensitivity from the reduced sample volumes. Here, we demonstrate that the sensitivity penalty is less severe than expected for highly paramagnetic solids and is more than offset by the associated improved resolution. While previous approaches employing slower MAS are often unsuccessful in providing sufficient resolution, we show that ultra‐fast 100+kHz MAS allows site‐specific assignments of all resonances from complex paramagnetic solids. Combined with more reliable rotor materials and handling methods, this opens the way to the routine characterization of geometry and electronic structures of functional paramagnetic systems in chemistry, including catalysts and battery materials. We benchmark this approach on a hygroscopic luminescent Tb3+ complex, an air‐sensitive homogeneous high‐spin Fe2+ catalyst, and a series of mixed Fe2+/Mn2+/Mg2+ olivine‐type cathode materials.

Resolving Structures of Paramagnetic Systems in Chemistry and Materials Science by Solid-State NMR: The Revolving Power of Ultra-Fast MAS.

Ultra-fast magic-angle spinning (100+kHz) has revolutionized solid-state NMR of biomolecular systems but has so far failed to gain ground for the analysis of paramagnetic organic and inorganic powders, despite the potential rewards from substantially improved spectral resolution. The principal blockages are that the smaller fast-spinning rotors present significant barriers for sample preparation, particularly for air/moisture-sensitive systems, and are associated with low sensitivity from the reduced sample volumes. Here, we demonstrate that the sensitivity penalty is less severe than expected for highly paramagnetic solids and is more than offset by the associated improved resolution. While previous approaches employing slower MAS are often unsuccessful in providing sufficient resolution, we show that ultra-fast 100+kHz MAS allows site-specific assignments of all resonances from complex paramagnetic solids. Combined with more reliable rotor materials and handling methods, this opens the way to the routine characterization of geometry and electronic structures of functional paramagnetic systems in chemistry, including catalysts and battery materials. We benchmark this approach on a hygroscopic luminescent Tb3+ complex, an air-sensitive homogeneous high-spin Fe2+ catalyst, and a series of mixed Fe2+/Mn2+/Mg2+ olivine-type cathode materials.

Design of an innovative aptasensor for the detection of chemotherapeutic drug Fludarabine phosphate

Monitoring the concentration of Fludarabine phosphate, a standard chemotherapeutic drug widely used in cancer treatment, is vital for ensuring the drug’s safety and effectiveness, tailoring treatments to individual needs, and consequently improving overall patient outcomes. Regarding the limitations of conventional techniques in terms of complexity, large time measurements, and a high cost, there is an urgent need to develop simple, rapid, and cost-effective devices. In this paper, we report the design of an aptasensor for the specific and selective detection of Fludarabine. Systematic evolution of ligands by exponential enrichment (SELEX) protocol was performed to select a specific aptamer for Fludarabine. Eleven rounds were carried out, and nine sequences were selected. Based on the dissociation constant (Kd), Ful-3, exhibiting the highest affinity (18.86 nM), was chosen and integrated into a simple electrochemical aptasensing platform for Fludarabine detection. Electrochemical results demonstrated good performance of the selected aptamer in detecting Fludarabine within the analytical range of 1 to 150 pg/mL and with LOD and LOQ in the order of 0.11 pg/mL (0.31 fM) and 0.39 pg/mL (1.06 fM), respectively. The developed platform also showed good selectivity against different analogous molecules and high applicability in serum-spiked samples, with recovery percentages ranging from 96.81 to 99.04%. Considering the encouraging results, this research presents an excellent alternative in terms of simplicity, stability, ease of use, reduction of sample volume, and low cost.

B-013 Improving the Workflow of Blood and Coagulation Tests in clinical laboratory through the Utilization of Laboratory Automation Track Systems

Abstract Background The Laboratory Automation Track System (TLA) is an automated system designed for executing highly repetitive tasks within a laboratory, commonly applied in biochemical and immunological testing systems. It aims to reduce human errors in specimen handling, enhance the overall process quality, and provide better Turnaround Time (TAT) from specimen collection to result reporting. For the first time in our laboratory, we have integrated the blood and coagulation systems with TLA. Consequently, we seek to evaluate the differences in the testing workflow and reporting efficiency of the blood and coagulation systems after being connected to TLA. Methods Abbott GLP systems Track was the TLA system for connecting with blood and coagulation systems. Two Sysmex XN-9100 fully automatic hematology systems and two Sysmex CS-5100 fully automatic coagulation analyzers were developed for blood and coagulation system, respectively. TUNYEN Laboratory Information System was established for Laboratory information system (LIS). Results Through the analysis of quality indicators and integration with intelligent information systems, improvements have been made, including the reduction of blood collection tube usage, efficient manpower utilization management, tracking of specimen progress and achievement of timely reporting rates.(1)Approximately 2,915 blood collection test tubes are saved every month, with a converted cost of 0.15 USD per tube, and a total saving of 437 USD. And reduce the carbon emissions of 32 kgCO2e, comply with the spirit of ESG sustainable development.(2)The TLA no longer requires staff to collect and deliver specimens as in the past,reducing staff and enhancing work efficiency.One of manpower was saved and new inspection items were developed.(3)Tracking of specimen progress:The laboratory has set up a specimen tracking system to monitor the specimen delivery location and provide timely response to the clinical status.The one-hour TAT for blood and coagulation increased from 82.0% and 76.9% before improvement to 91.8% and 93.3% after improvement respectively. Conclusions The implementation of Laboratory Automation Track Systems (TLA) leads to a reduction in medical errors and specimen sample volume, while also enhancing accuracy and precision, thereby improving the safety of laboratory staff. Although the introduction of TLA may require an initial investment, it brings the benefits of automation, offering ease and efficiency to laboratory processes.

Development of two ultra-sensitive UHPLC-QqQ-MS/MS methods for the simultaneous determination of hydroxyzine and its active metabolite (cetirizine) in human blood: applications to real cases of forensic toxicology.

Both postmortem toxicological and medical-forensic examinations are very important in the case of analyzing various types of chemical substances. Hydroxyzine (HZ) is a first-generation antihistamine drug with a sedative effect that disrupts cognitive function and affects the ability to drive motor vehicles. Enzymatic oxidation of the hydroxy-methyl group to the carboxyl group leads to the formation of its main metabolite-cetirizine (CZ). CZ is the active substance of antiallergic drugs. Because it does not cross the BBB (blood-brain barrier) easily, it is less likely to cause drowsiness or affect memory and impair cognitive function. Therefore, in criminal studies, it is often important what medication had been taken by a person involved, e.g., in a car accident, HZ or CZ. The analysis of both antihistamine drugs is challenging, as usually very low concentrations of the compound of interest need to be determined. Thus, an ultra-sensitive UHPLC-QqQ-MS/MS method was developed for simultaneous determination of HZ and CZ in biological fluid samples. The lower limit of quantification (LOQ) for HZ and CZ was calculated as 0.345 and 0.3696ng/mL, respectively. Together with a reduced sample volume to 200 μL, it makes the developed method suitable for a sensitive multidrug forensic toxicological analysis. Samples were extracted with simple and fast liquid-liquid extraction (ethyl acetate, pH 9). The present method for the determination of HZ and CZ in human blood proved to be simple, fast, selective, and sensitive. The quantification by LC-MS/MS was successfully applied to the samples coming from 28 authentic biological fluids (blood, urine, vitreous humor, bile and stomach content), both antemortem and postmortem. The performed studies confirm that the developed method is characterized by a high extraction efficiency. Its accuracy, reproducibility, simplicity, and selectivity suggest its application in clinical, toxicological, and forensic laboratories.

Three‐Dimensional Printed Integrated Electrochemical Devices for Various Applications–A Review

AbstractThree‐dimensional Printing (3DP) and computer‐assisted design (CAD) are a boon for producing high‐quality analytical and electrochemical devices using low‐cost components. This review briefly explains various 3D printing techniques. The focus is on the fabrication of integrated miniature devices developed through the 3DP process, mainly by the Fused deposition modelling (FDM) technique. Examples of integrated electrochemical devices are presented to highlight the potential of 3DP. Special emphasis is given to surface activation of 3D printed electrodes to enhance their electrochemical activity and various activation methods. This paper discusses the opportunities and future applications in developing all‐in‐one miniature electrochemical devices that might lead to on‐site environmental measurements, point‐of‐care tests, and the development of portable instruments with reduced sample volume.

Glass capillary systems for micro-volume fluorometry

Capillary-based fluorometry can be considered a promising alternative to cuvette-based methods, enabling low-volume measurements while reducing analyte consumption and waste generation. In this work, we compared the performance of three types of glass container systems, i.e., anti-resonant hollow core fibers, capillaries, and standard cuvettes, using green fluorescent protein (GFP) as a reference analyte. Among these, when the volume of the analyte and the fluorescence intensity are assessed, a capillary accommodating 2.9 μL was found as an optimal choice. The capillary-based interrogation setup was further optimized to enhance sensitivity by adjusting the florescence-exciting laser beam position and optimizing the capillary’s shape. The obtained capillary-based setup achieved a noteworthy 85 % reduction in sample volume compared to previous studies utilizing capillary-based fluorometry, highlighting its significant contribution to material conservation. The system achieved a limit of detection as low as 26 μg/mL (1.01 nM) GFP concentration and a resolution of 3.5 μg/mL. With its low cost and potential for reusability, capillary-based fluorometry presents a compelling alternative to traditional cuvette-based fluorometry, offering a promising solution for micro-volume analysis.

Ten Thousand Recaptures of a Single DNA Molecule in a Nanopore and Variance Reduction of Translocation Characteristics.

Nanopores have emerged as highly sensitive biosensors operating at the single-molecule level. However, the majority of nanopore experiments still rely on averaging signals from multiple molecules, introducing systematic errors. To overcome this limitation and obtain accurate information from a single molecule, the molecular ping-pong methodology provides a precise approach involving repeated captures of a single molecule. In this study, we have enhanced the molecular ping-pong technique by incorporating a customized electronic system and control algorithm, resulting in a recapture number exceeding 10,000. During the ping-pong process, we observed a significant reduction in the variance of translocation characteristics, providing fresh insights into single-molecule translocation dynamics. An inhomogeneous translocation velocity of folded DNA has been revealed, illustrating a strong interaction between the molecule and the solid-state nanopore. The results not only promise heightened experimental efficiency with reduced sample volume but also increase the precision in statistical analysis of translocation events, marking a significant stride toward authentic single-molecule nanopore sensing.

Optimized waveguides for mid-infrared lab-on-chip systems: A rigorous design approach

Mid-infrared absorption spectroscopy is a well-established technique for non-destructive quantitative molecular analysis. Waveguide-integrated sensors provide a particularly compact solution operating with reduced sample volumes while exhibiting exquisite molecular selectivity, sensitivity, and ultra-low limits of detection. Recent advances in mid-infrared technologies along with the integration of on-chip sources, detectors and microfluidics, have brought mid-infrared lab-on-chip systems closer to reality. A variety of material platforms has been proposed for the implementation of such systems. However, the lack of a consistent waveguide design approach renders a fair comparison between different alternatives – and a deliberate material selection – challenging, limiting the development of optimized on-chip spectroscopic devices. In the present study, a systematic waveguide design approach has been developed, facilitating evanescent field absorption-based sensing, in particular for aqueous analytes. Our strategy enables a rigorous comparison of several state-of-the-art thin-film waveguides using parametric expressions to predict the achievable limits of detection of the sensing system, while indicating optimum waveguide dimensions and absorption pathlengths, pivotal for the development of next-generation mid-infrared lab-on-chip devices.

English

In the last years, the use of Dried Blood Spots (DBS) as alternative sampling technique is reaching an increasing interest in the antidoping field. The collection of DBS is performed from small volumes of capillary blood lied on adsorbent support (e.g.cellulose) and let to dry. Compared to the conventional urine or blood collection, this technique presents several advantages as the sampling is less invasive, rapid and does not required specialized personnel to be performed. It also facilitates transport and storage, reducing the costs. Conversely, the reduced sample volume available could represent a limitation and requires the use of highly sensitive instrumentation. This work presents a multi-class screening method for 100 compounds belonging to different groups of the World Anti Doping Agency prohibited list, including anabolic agents, beta-2 agonists, hormone and metabolic modulators, and diuretics, among others. DBS samples for method development were obtained depositing 20 µL of venous blood and letting them to dry on cellulose cards. The whole DBS spot was then punched out and extracted with organic solvents prior to the analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). The methodology was validated for qualitative purposes. Different parameters were evaluated. The vast majority of the compounds could be reliably detected at sub-ng/mL level. Satisfactory results were obtained in terms of recovery, precision and robustness. Also, matrix effects were negligible for most compounds, as expected considering the low volume of sample analyzed. As a final validation step, DBS samples collected after administration of boldenone, oxandrolone and tamoxifen to healthy volunteers were analyzed. The method showed good performance and robust results, making it fit-for- purpose for its application in sports drug testing.

Enhancing drug development and clinical studies with patient-centric sampling using microsampling techniques: Opportunities, challenges, and insights into liquid chromatography-mass spectrometrystrategies.

Microsampling has revolutionized pharmaceutical drug development and clinical research by reducing sample volume requirements, allowing sample collection at home or nontraditional sites, minimizing animal and patient burden, and enabling more flexible study designs. This perspective paper discusses the transformative impact of microsampling and patient-centric sampling (PCS) techniques, emphasizing their advantages in drug development and clinical trials. We highlight the integration of liquid chromatography-mass spectrometry (LC-MS) strategies for analyzing PCS samples, focusing on our research experience and a review of current literatures. The paper reviews commercially available PCS devices, their regulatory status, and their application in clinical trials, underscoring the benefits of PCS in expanding patient enrollment diversity and improving study designs. We also address the operational challenges of implementing PCS, including the need for bridging studies to ensure data comparability between traditional and microsampling methods, and the analytical challenges posed by PCS samples. The paper proposes future directions for PCS, including the development of global regulatory standards, technological advancements to enhance user experience, the increased concern of sustainability and patient data privacy, and the integration of PCS with other technologies for improved performance in drug development and clinical studies. By advancing microsampling and PCS techniques, we aim to foster patient-centric approaches in pharmaceutical sciences, ultimately enhancing patient care and treatment efficacy.

Miniaturized on-spot protein denaturation/microwave-assisted extraction for spectrofluorimetric determination of favipiravir in dried plasma: Application to real human sample and inclusive incurred sample reanalysis study

The development of high-throughput sample preparation methods is a key step for upgrading the throughput of classical analytical techniques. In this work, a miniaturized on-spot protein denaturation coupled with microwave-assisted extraction sample treatment method was developed for extraction and subsequent spectrofluorimetric determination of favipiravir (FAV) in human plasma spots. Dried plasma spotting was performed using only 10 µL of plasma on filter paper discs followed by on-spot protein denaturation using 10 µL methanol. Maximum extraction efficiency was achieved using water: methanol (1:1) as extraction solvent with 50 % microwave power for 60 sec. The fluorescence intensity of extracted FAV was measured at 432 nm after excitation at 361 nm. According to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH -M10) guidelines for bioanalytical method validation, spiked plasma samples showed very good linearity between 100 and 1000 ng/mL. The accuracy and precision of the developed method were indicated by mean recovery (%) within 100 ± 15 % for all tested concentrations levels. The obtained results were further compared with a reported protein precipitation method using both F-test and t-test revealing no significant difference between either method. Moreover, the method was successfully applied for FAV determination in real human samples for an incurred sample reanalysis study revealing no metabolite back conversion. The developed method showed superior green performance using analytical Eco-scale, Green Analytical Procedure Index (GAPI), and Analytical Greenness Metric Approach (AGREE) tools compared to a classical protein precipitation method. According to Blue Applicability Grade Index (BAGI), and whiteness tools, the developed method outperformed all reported methods for FAV determination in plasma in terms of practicality and analytical functionality. Compared to reported sample treatment methods, this method exhibited excellent analytical performance, increased throughput, hundred-fold reduction of sample volume (10 μL), significant reduction of organic solvent consumption (260 μL methanol) and produced negligible wastes. Thus, it can be adopted for routine work in pharmacokinetics and clinical trial studies.