UPLC-MS/MS method for fluconazole determination in plasma and its application in Mexican patients with candidaemia.

Aim: An accurate and fast ultra-high performance liquid chromatography coupled with tandem mass spectrometry analytical method was developed and validated for quantifying fluconazole levels in human plasma according to the US FDA guidelines.Materials & methods: A simple protein precipitation by acetonitrile was employed for the sample preparation. The chromatographic separation was carried out using isocratic elution of water (0.1% formic acid) and acetonitrile on an Acquity ultra-high performance liquid chromatography HSS T3 column. Samples from ten adult patients diagnosed with candidemia who received fluconazole treatment were analyzed.Results & conclusion: The method demonstrated excellent linearity and stability within the 1-50 µg/ml range (r2 >0.999). The intraday and interday precision were determined with coefficient of variation values ranging from 1.4 to 4.38% and 2.8 to 6.6%, respectively. This rapid and selective method has successfully analyzed 27 plasma samples. The straightforward sample preparation in a single step and the reduced analysis time make this method suitable for adult patients with candidemia, leading to improved clinical outcomes.

[Box: see text].

Development of a sensitive LC-MS/MS assay to support human microdose study for an oral agonist of the GLP-1 receptor.

Aim: The purpose of this work was to determine the feasibility of supporting a clinical microdose study for PF-06882961 (danuglipron), an oral small molecule agonist of the GLP-1 receptor, by LC-MS/MS. Methodology: Statistical instrument parameter optimization using response surface methodology was employed to develop a LC-MS/MS method for the analyte, PF-06882961. Results: An LC-MS/MS method was developed and validated to support a proof of concept microdose pharmacokinetics preclinical study in monkeys, administered PF-06882961 (0.005 mg total, average dose = 0.0007 mg/kg) via intravenous bolus injection. Conclusion: The present study demonstrated the feasibility of analyzing human microdose plasma samples for PF-06882961 by LC-MS/MS, instead of accelerator mass spectrometry, thereby reducing cost and eliminating synthesis and exposure to 14C labeled material.

[Box: see text].

Current green capillary electrophoresis and liquid chromatography methods for analysis of pharmaceutical and biomedical samples (2019-2023) - A review.

Separation analytical methods, including liquid chromatography (LC) and capillary electrophoresis (CE), in combination with an appropriate detection technique, are dominant and powerful approaches preferred in the analysis of pharmaceutical and biomedical samples. Recent trends in analytical methods are focused on activities that push them to the field of greenness and sustainability. New approaches based on the implementation of greener solvents, non-hazardous chemicals, and reagents have grown exponentially. Similarly, recent trends are pushed in to the strategies based on miniaturization, reduction of wastes, avoiding derivatization procedures, or reduction of energy consumption. However, the real greenness of the analytical method can be evaluated only according to an objective and sufficient metric offering complex results taking into account all twelve rules of green analytical chemistry (SIGNIFICANCE mnemonic system). This review provides an extensive overview of papers published in the area of development of green LC and CE methods in the field of pharmaceutical and biomedical analysis over the last 5 years (2019-2023). The main focus is situated on the metrics used for greenness evaluation of the methods applied for the determination of bioactive agents. It critically evaluates and compares the demands of the real applicability of the methods in quality control and clinical environment with the requirements of the green analytical chemistry (GAC). Greenness and practicality of the summarized methods are re-evaluated or newly evaluated with the use of the dominant metrics tools, i.e., Analytical GREEnness (AGREE), Green Analytical Procedure Index (GAPI), Blue Applicability Grade Index (BAGI), and Sample Preparation Metric of Sustainability (SPMS). Moreover, general conclusions and future perspectives of the greening procedures and greenness evaluation metrics systems are presented. This paper should provide comprehensive information to analytical chemists, biochemists, and it can also represent a valuable source of information for clinicians, biomedical or quality control laboratories interested in development of analytical methods based on greenness, practicality, and sustainability.

Techniques for characterizing biofunctionalized surfaces for bioanalysis purposes.

Surface biofunctionalization is an essential stage in the preparation of any bioassay affecting its analytical performance. However, a complete characterization of the biofunctionalized surface, considering studies of coverage density, distribution and orientation of biomolecules, layer thickness, and target biorecognition efficiency, is not met most of the time. This review is a critical overview of the main techniques and strategies used for characterizing biofunctionalized surfaces and the process in between. Emphasis is given to scanning force microscopies as the most versatile and suitable tools to evaluate the quality of the biofunctionalized surfaces in real-time dynamic experiments, highlighting the helpful of atomic force microscopy, Kelvin probe force microscopy, electrochemical atomic force microscopy and photo-induced force microscopy. Other techniques such as optical and electronic microscopies, quartz crystal microbalance, X-ray photoelectron spectroscopy, contact angle, and electrochemical techniques, are also discussed regarding their advantages and disadvantages in addressing the whole characterization of the biomodified surface. Scarce reviews point out the importance of practicing an entire characterization of the biofunctionalized surfaces. This is the first review that embraces this topic discussing a wide variety of characterization tools applied in any bioanalysis platform developed to detect both clinical and environmental analytes. This survey provides information to the analysts on the applications, strengths, and weaknesses of the techniques discussed here to extract fruitful insights from them. The aim is to prompt and help the analysts to accomplish an entire assessment of the biofunctionalized surface, and profit from the information obtained to enhance the bioanalysis output.

Analysis of indoxyl sulfate in biological fluids with emphasis on sample preparation techniques: A comprehensive analytical review.

The uremic toxin indoxyl sulfate (IS) has been related to the development of various medical conditions notably chronic kidney disease (CKD). Hence, quantification of this biomarker in biological fluids may be a diagnostic tool to evaluate renal system functionality. Numerous analytical methods including liquid chromatography, gas chromatography, spectroscopy, and electrochemical techniques have since been used to analyze IS in different biological fluids. The current review highlights the relevant studies that assessed IS with a special focus on sample preparation, which is essential to reduce or eliminate the effect of endogenous components from the matrix in bioanalysis.

LC-MS/MS-based bioanalysis of branched-chain and aromatic amino acids in human serum.

Aim: Branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) were suggested as potential biomarkers in liver disease. This study aimed to develop and validate a simple and rapid LC-MS/MS method to simultaneously measure serum BCAAs and AAAs levels in patients with liver injury, and further establish reference intervals of Chinese healthy adult populations.Patients & methods: Samples were prepared by a one-step protein precipitation and analysis time was 4 min per run.Results: The validation results showed good linearity (r2 >0.9969), satisfactory accuracy (94.44% - 107.75%) and precision (0.10% - 5.90%).Conclusion: This method proved to be suitable for high-throughput routine clinical use and could be a valuable adjunct diagnosis tool for liver injury and other clinical applications.

[Box: see text].

Analysis of human biological samples using porous graphitic carbon columns and liquid chromatography-mass spectrometry: a review.

Liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful analytical technique for analyzing complex biological samples. Among various chromatographic stationary phases, porous graphitic carbon (PGC) columns have attracted significant attention due to their unique properties-such as the ability to separate both polar and non-polar compounds and their stability through all pH ranges and to high temperatures-besides the compatibility with LC-MS. This review discusses the applicability of PGC for SPE and separation in LC-MS-based analyses of human biological samples, highlighting the diverse applications of PGC-LC-MS in analyzing endogenous metabolites, pharmaceuticals, and biomarkers, such as glycans, proteins, oligosaccharides, sugar phosphates, and nucleotides. Additionally, the fundamental principles underlying PGC column chemistry and its advantages, challenges, and advances in method development are explored. This comprehensive review aims to provide researchers and practitioners with a valuable resource for understanding the capabilities and limitations of PGC columns in LC-MS-based analysis of human biological samples, thereby facilitating advancements in analytical methodologies and biomedical research.

Quantitative determination of liposomal irinotecan and SN-38 concentrations in plasma samples from children with solid tumors: Use of a cryoprotectant solution to enhance liposome stability.

Preclinical studies have demonstrated that liposomal irinotecan (CPT-11), a topoisomerase I inhibitor, has broad activity against adult cancers, including pancreatic, gastric, colon, lung, glioma, ovarian, and breast cancer. Encapsulation of irinotecan into liposomes can modify its pharmacokinetic properties dramatically. Also, the pharmacokinetic profiles of liposomal drug formulations are not fully understood; thus, bioanalytical methods are needed to separate and quantify nonencapsulated vs. encapsulated concentrations. In this study, two robust, specific, and sensitive LC-MS/MS methods were developed and validated to separate and quantify the nonencapsulated CPT-11 (NE-CPT-11) from the sum-total CPT-11 (T-CPT-11) and its major metabolite, SN-38, in human plasma after intravenous administration of liposomal irinotecan. NE-CPT-11 and SN-38 were separated from plasma samples by using solid-phase extraction, and T-CPT-11 was measured by protein precipitation. The liposomal CPT-11 formulation was unstable during sample storage and handling, resulting in elevated NE-CPT-11 concentration. To improve the stability of liposomal CPT-11, a cryoprotectant solution was added to human plasma samples prior to storage and processing. CPT-11, SN-38, and their respective internal standards, CPT-11-d10 and SN-38-d3, were chromatographically separated on a reversed-phase C18 analytical column. The drugs were detected on a triple quadrupole mass spectrometer in the positive MRM ion mode by monitoring the transitions 587.3 > 124.1 (CPT-11) and 393.0 > 349.1 (SN-38). The calibration curves demonstrated a good fit across the concentration ranges of 10-5000 ng/mL for T-CPT-11, 2.5-250 ng/mL for NE-CPT-11, and 1-500 ng/mL for SN-38. The accuracy and precision were within the acceptable limits, matrix effects were nonsignificant, recoveries were consistent and reproducible, and the analytes were stable under all tested storage conditions. Finally, the LC-MS/MS methods were successfully applied in a phase I clinical pharmacokinetic study of nanoliposomal irinotecan (Onivyde®) in pediatric patients with recurrent solid malignancies or Ewing sarcoma.

Validation of a method for the determination of Aderamastat (FP-025) in K2EDTA human plasma by LC-MS/MS.

Aderamastat (FP-025) is a small molecule, selective matrix metalloproteinase (MMP)-12 inhibitor, under development for respiratory conditions which may include chronic inflammatory airway diseases and pulmonary fibrosis. To support evaluation of the pharmacokinetic parameters of Aderamastat in humans, we developed and validated a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical method for the quantification of Aderamastat in human plasma. This assay was validated in compliance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations (GLP) and European Medicines Agency (EMA) guidelines. K2EDTA human plasma samples were spiked with internal standard, processed by liquid-liquid extraction, and analyzed using reversed-phase HPLC with Turbo Ion Spray® MS/MS detection. Separation was done using a chromatographic gradient on 5 µm C6-Phenyl 110 Å, 50*2 mm analytical column at a temperature of 35 °C. The LC-MS/MS bioanalytical method, developed by QPS Taiwan to determine the concentration of Aderamastat in K2EDTA human plasma, was successfully validated with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, lipemic plasma, batch size, recovery, matrix effect, and carry-over. These data indicate that the method for determination of Aderamastat concentrations in human K2EDTA plasma can be used in pharmacokinetics studies and subsequent clinical trials with Aderamastat. Authors declare that, this novel data is not published and not under consideration for publication by another journal than this journal. All data will be made available on request.

Microbore-based vacuum jacketed liquid chromatography for fast, efficient and sustainable high throughput bioanalysis.

Extra-column band broadening can significantly reduce the performance of rapid ultra-high performance liquid chromatography-MS-based methods (UHPLC-MS). However, as we show here, UHPLC-MS/MS methods on short 2.1 mm i.d. columns can be optimized to reduce band broadening by simple procedures such as dispensing with the solvent divert valves placed between the column and the MS source. Vacuum jacketed columns have previously been shown to provide superior performance to conventional UHPLC-MS/MS by reducing on and post column band broadening. Here we have compared the optimized "direct" UHPLC approach for the high throughput (HT) bioanalysis of drugs and metabolites in biofluids such as urine and blood plasma with vacuum jacketed chromatography (VJC), using columns of the same geometry and packed with the same stationary phases. This study demonstrates that the performance of VJC was still superior to the direct UHPLC-MS/MS methods for rapid "generic" bioanalysis using gradient times of 0.25 to 5 min. Further investigations using microbore VJC-MS/MS, with 1 mm i.d. columns, for bioanalysis of the same biofluid samples showed that this format offers great promise for HT "discovery" drug and metabolite analysis/profiling. In addition the reduction of solvent use, by up to 90 % for methods when using microbore columns, can significantly contribute to improved sustainability and reducing costs per analysis.

Capillary mediated vitrification is a novel technique that enables storage of antibody critical reagents at ambient temperature: Impact on binding, structure, and laboratory sustainability.

Antibodies and antibody conjugates are essential components of life science research, but their inherent instability necessitates cold storage or lyophilization, posing logistical and sustainability challenges. Capillary-mediated vitrification has shown promise as a tool for improving biomolecule stability. In this study, we assess the feasibility of shipping and storing CMV-stabilized antibody reagents at ambient temperature using a purified rabbit polyclonal as a model system. The conditions tested included a simulated temperature excursion, ambient shipping, and storage for approximately two months at room-temperature. Antibody function was measured by both ELISA and Octet bio-layer interferometry kinetic measurements. Yield, aggregation, and thermal stability were assessed by UV/VIS, Size Exclusion Chromatography (SEC), thermal melting, and thermal aggregation studies. Results indicate >97 % protein yield and no impact on the binding activity. No evidence of aggregation or oligomer formation was detected. Addition of the vitrification buffer to the sample matrix resulted in an increase in the aggregation on-set temperature, indicating enhanced thermostability. A slight shift in both the SEC retention time for the main peak and a difference in aggregation behavior at high temperatures were noted post-vitrification. We hypothesize that these differences are related to the interaction of the protein with the saccharide component of the vitrification matrix and the stabilization mechanism of sugars. The cumulative data supports the use of Capillary Mediated Vitrification as a viable alternative to frozen reagent storage, with the potential to significantly impact reagent stability, assay performance, laboratory operations, and sustainability initiatives.

Advances in analytical technologies for emerging drug modalities and their separation challenges in LC-MS systems.

The last few years have seen a rise in the identification and development of bio-therapeutics through the use of cutting-edge delivery methods or bio-formulations, which has created bio-analytical difficulties. Every year, new bio-pharmaceutical product innovations come out, but the analytical development of these products is challenging. Quantifying the products and components of conjugated molecular structures is essential for preclinical and clinical research in order to guide therapeutic development, given their intrinsic complexity. Furthermore, a significant amount of information is needed for the measurement of these unique modalities by LC-MS techniques. Numerous LC-MS based methods have been developed, including AEX-HPLC-MS, RP-IP-LCMS, HILIC-MS, LCHRMS, Microflow-LC-MS, ASMS, Hybrid LBA/LC-MS, and more. However, these methods continue to face problems, prompting the development of alternative approaches. Therefore, developing bio-molecules that are this complicated and, low in concentration requires a skilled LC-MS based approach and knowledgeable personnel. This review covers general novel modalities classifications, sample preparation techniques, current status and bio-analytical strategies for analyzing various novel modalities, including gene bio-therapeutics, oligonucleotides, antibody-drug conjugates, monoclonal antibodies and PROTACs. It also covers how these strategies have been used in the past and how they are being used now to address challenges in the development of LC-MS based methods, as well as improvement strategies, current advancements and recent developed methods. We additionally covered on the benefits and drawbacks of different LC-MS based techniques for the examination of bio-pharmaceutical products and the future perspectives.

An Update on Current Trend in Sample Preparation Automation in Bioanalysis: strategies, Challenges and Future Direction.

Automation in sample preparation improves accuracy, productivity, and precision in bioanalysis. Moreover, it reduces resource consumption for repetitive procedures. Automated sample analysis allows uninterrupted handling of large volumes of biological samples originating from preclinical and clinical studies. Automation significantly helps in management of complex testing methods where generation of large volumes of data is required for process monitoring. Compared to traditional sample preparation processes, automated procedures reduce associated expenses and manual error, facilitate laboratory transfers, enhance data quality, and better protect the health of analysts. Automated sample preparation techniques based on robotics potentially increase the throughput of bioanalytical laboratories. Robotic liquid handler, an automated sample preparation system built on a robotic technique ensures optimal laboratory output while saving expensive solvents, manpower, and time. Nowadays, most of the traditional extraction processes are being automated using several formats of online techniques. This review covered most of the automated sample preparation techniques reported till date, which accelerated and simplified the sample preparation procedure for bioanalytical sample analysis. This article critically analyzed different developmental aspects of automated sample preparation techniques based on robotics as well as conventional sample preparation methods that are accelerated using automated technologies.

Hyphenation of microflow chromatography with electrospray ionization mass spectrometry for bioanalytical applications focusing on low molecular weight compounds: A tutorial review.

Benefits of miniaturized chromatography with various detection modes, such as increased sensitivity, chromatographic efficiency, and speed, were recognized nearly 50 years ago. Over the past two decades, this approach has experienced rapid growth, driven by the emergence of mass spectrometry applications serving -omics sciences and the need for analyzing minute volumes of precious samples with ever higher sensitivity. While nanoscale liquid chromatography (flow rates <1 µL/min) has gained widespread recognition in proteomics, the adoption of microscale setups (flow rates ranging from 1 to 100 µL/min) for low molecular weight compound applications, including metabolomics, has been surprisingly slow, despite the inherent advantages of the approach. Highly heterogeneous matrices and chemical structures accompanied by a relative lack of options for both selective sample preparation and user-friendly equipment are usually reported as major hindrances. To facilitate the wider implementation of microscale analyses, we present here a comprehensive tutorial encompassing important theoretical and practical considerations. We provide fundamental principles in micro-chromatography and guide the reader through the main elements of a microflow workflow, from LC pumps to ionization devices. Finally, based on both our literature overview and experience, illustrated by some in-house data, we highlight the critical importance of the ionization source design and its careful optimization to achieve significant sensitivity improvement.

Bacteriophage-Based Bioanalysis.

Bacteriophages, which are viral predators of bacteria, have evolved to efficiently recognize, bind, infect, and lyse their host, resulting in the release of tens to hundreds of propagated viruses. These abilities have attracted biosensor developers who have developed new methods to detect bacteria. Recently, several comprehensive reviews have covered many of the advances made regarding the performance of phage-based biosensors. Therefore, in this review, we first describe the landscape of phage-based biosensors and then cover advances in other aspects of phage biology and engineering that can be used to make high-impact contributions to biosensor development. Many of these advances are in fields adjacent to analytical chemistry such as synthetic biology, machine learning, and genetic engineering and will allow those looking to develop phage-based biosensors to start taking alternative approaches, such as a bottom-up design and synthesis of custom phages with the singular task of detecting their host.

Bioanalysis of free antisense oligonucleotide payload from antibody-oligonucleotide conjugate by hybridization LC-MS/MS.

Background: Antisense oligonucleotides (ASOs) have been conjugated to various moieties, such as peptides, antibodies or Fab regions of antibodies, to enhance their delivery to target tissues. The quantitation of free ASO (ASO payload) is critical to characterize its pharmacokinetics/pharmacodynamics (PK/PD) properties and biodistribution after delivery of the peptide/antibody/Fab ASO conjugates. Results: We developed a hybridization-based LC-MS/MS methodology for quantification of free ASO in tissues in the presence of Fab-ASO and ASO with linker (ASO-linker). Conclusion: The developed method was applied to measure accurately the free ASO concentrations in liver and gastrocnemius in mice that were dosed with Fab-ASO. This methodology has also been applied to free ASO bioanalysis for other antibody-ASO and Fab-ASO conjugates in various tissues and plasma/serum samples.

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Biosensors and Bioassays for the Medical Detection of Bacillus Anthracis and Diagnosis of Anthrax.

Bacillus anthracis is a causative agent of the highly mortal disease anthrax. This zoonosis is present in nature, but it is also considered one of the most powerful biological warfare agents. A timely diagnosis is necessary for proper therapy and setting of epidemiological countermeasures. Current diagnostic methods should be used in specialized laboratories or medical facilities because there are only a limited number of methods suitable as hand-held assays or even point-of-care tests for detecting B. anthracis or anthrax diagnosis. The lateral flow tests are an exception in this regard, but these tests also have some limitations. Significant progress has been achieved in point-of-care tests for B. anthracis detection and anthrax diagnosis in various biosensors and bioassays. This review focuses on current hand-held and point-of-care tests that can easily prove anthrax or its causative agent outside the context of specialized facilities.

Artificial-Cofactor-Mediated Hydrogen and Electron Transfer Endows AuFe/Polydopamine Superparticles with Enhanced Glucose Oxidase-Like Activity.

Various applications related to glucose catalysis have led to the development of functional nanozymes with glucose oxidase (GOX)-like activity. However, the unsatisfactory catalytic activity of nanozymes is a major challenge for their practical applications due to their inefficient hydrogen and electron transfer. Herein, we present the synthesis of AuFe/polydopamine (PDA) superparticles that exhibit photothermal-enhanced GOX-like activity. Experimental investigations and theoretical calculations reveal that the glucose oxidation process catalyzed by AuFe/PDA follows an artificial-cofactor-mediated hydrogen atom transfer mechanism, which facilitates the generation of carbon-centered radical intermediates. Rather than depending on charged Au surfaces for thermodynamically unstable hydride transfer, Fe(III)-coordinated PDA with abundant amino and phenolic hydroxyl groups serves as cofactor mimics, facilitating both hydrogen atom and electron transfer in the catalytic process. Finally, leveraging the photothermal-enhanced GOX-like and catalase-like activities of AuFe/PDA, we establish a highly sensitive and accurate point-of-care testing blood glucose determination with exceptional anti-jamming capabilities.

Considerations and perspectives on the practice of incurred sample reanalysis assessment for chiral drugs.

Given the inherent complexities of bioanalysis, the role of incurred sample reanalysis (ISR) is increasingly appreciated in regulatory bioanalysis. Incurred sample reanalysis has evolved as an integral part of an assay to ensure method reproducibility. The current regulatory ISR guidelines do not provide clarity regarding ISR assessment for chiral drugs comprising enantiomers. Because chiral assays evaluate two enantiomers, there are additional complexities associated with the ISR data generation and interpretation. Based on the current literature, the practices for conducting ISR in chiral methods were reviewed and assessed. While ISR was conducted in chiral methods for both enantiomers using the acceptance criteria prescribed for non-chiral methods, there may be a need to streamline the nuances of ISR data interpretation and define the ISR requirements for chiral methods. The article provides perspectives on the ISR of enantiomeric drugs, including strategy development, by providing various hypothetical scenarios and possible considerations for defining ISR evaluation for chiral assays.