Clean and Complete Protein Digestion with an Autolysis Resistant Trypsin for Peptide Mapping.

Peptide mapping requires cleavage of proteins in a predictable fashion so that target protein-specific peptides can be reliably identified and quantified. Trypsin, a commonly used protease in this process, can also undergo self-cleavage or autolysis, thereby reducing the effectivity and even cleavage specificity at lysine and arginine residues. Here, we report highly efficient and reproducible peptide mapping of biotherapeutic monoclonal antibodies. We highlight the properties of a homogeneous chemically modified trypsin on thermal stability, a 54% increase in melting temperature with an 84% increase in energy required for unfolding, an indication of more thermally stable trypsin, >90% retained intact mass peak area after exposure to digestion conditions confirming autolysis resistance, 10× more intensity for intact enzyme compared to trypsin of similar source and narrower molecular weight distribution with LC-MS indicative of low degradation compared to 3 other types of trypsin. Finally, we show the utility of this autolysis-resistant trypsin in characterizing biotherapeutic monoclonal antibodies consistently and reliably showing a >30% reduction in missed cleavage for a short-duration protein digestion time of 30 min compared to heterogeneously modified trypsin of a similar source.

Redox interference-free bimodal paraoxon sensing enabled by an aggregation-induced emission nanozyme catalytically hydrolyzing phosphoesters specifically.

In view of the current serious situation of organophosphorus pesticides (OPs) residue contamination, developing rapid and accurate OPs sensors is a matter of urgency. Redox-nanozyme based colorimetric sensors have been widely researched and utilized in OPs residue determination, but overcoming the interference of external redox substances and the effect of single-signal modes on detection performance is still a challenge. Here we fabricated a Zr-based metal-organic framework (MOF) featuring specific phosphatase-like activity and strong aggregation-induced emission (AIE) fluorescence for redox interference-free bimodal pesticide sensing. In the MOF, the activity-tunable Zr4+ node offered high hydrolytic activity and affinity toward P-O containing substrates, and the rigid framework structure effectively enhanced the fluorescence emission of the ligand 1,1,2,2-tetra(4-carboxylphenyl)ethylene. The developed AIEzyme could efficiently catalyze the hydrolysis of paraoxon to yellow p-nitrophenol, which further reduced the intrinsic AIE fluorescence of AIEzyme through internal filtration effect. Thereby, a natural enzyme-free dual-mode colorimetric/fluorescence approach was established for paraoxon detection with no interference from redox substances, and a smartphone-assisted portable platform was further developed to enable the facile, rapid, and high-performance sensing of the pesticide in complex practical matrices.

Ocular pharmacokinetics of acetazolamide from intravitreal implants by high-performance liquid chromatography coupled to tandem mass spectrometry.

Glaucoma, a leading cause of irreversible blindness, affects about 70 million people globally. Its treatment focuses on reducing intraocular pressure. Acetazolamide, a potent anti-glaucoma drug, is currently used only systemically due to low solubility and permeation, which cause severe side effects. Developing topical medications with acetazolamide requires robust analytical methods for its detection in biological samples. In this context, this study aimed to develop a method to quantify acetazolamide in rabbit vitreous humor samples. The method involved a simple, fast, inexpensive, and environmentally friendly protein precipitation step for sample preparation, needing just 50 µL of sample and 200 µL of organic solvent, with adequate recovery. This was combined with high-performance liquid chromatography coupled to tandem mass spectrometry, enabling highly sensitive (LOQ of 5 ng/mL) quantification within only 5 min. The method proved to be selective, precise, and accurate, with well-fitted analytical curves, with no carryover, and no matrix effect impacting reliability. The method was successfully applied to analyze vitreous humor samples from rabbits in pharmacokinetic studies, monitoring drug release from intravitreal implants. Results showed a controlled release profile, with a maximum drug concentration (Cmax) of 426.01 ± 64.57 ng/mL, time to reach Cmax (Tmax) of 28 days, and area under the curve (AUC0-42 and AUC0-∞) of 7722.66 ± 1125.96 ng days/mL and 8998.11 ± 1311.92 ng days/mL, respectively. The device demonstrated significantly slower elimination, ensuring therapeutic levels for an extended period when compared to intravitreal injection.

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.

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An all-in-one smartphone-assisted ratiometric fluorescent device for visual and quantitative detection of glutathione.

The daily consumption of foods abundant in Glutathione (GSH) can be supplemented to maintain the homeostasis of GSH in human health and alleviate pathologies resulting from abnormal GSH levels. The fluorescence-based visual determination of GSH has gradually attracted the attention of researchers due to its robust performance and versatile implementation. However, the current GSH visual strategy primarily relies on variations in fluorescence intensity at a single emission wavelength, which poses challenges for naked-eye and portable readout, as well as distorted signals caused by complex matrix effects in real samples. Herein, a ratiometric fluorescence sensor based on carbon dots (CDs) combined with an all-in-one 3D-printed smartphone-based device was successfully developed for low-cost, visual and rapid detection of GSH without the need for an external excitation light source. The ratiometric fluorescent materials were synthesized by conjugating blue carbon dots (B-CDs) with yellow carbon dots (Y-CDs) through the utilization of selected Cu2+ ions. The resulting mechanism demonstrated that the coordination interaction between Cu2+ and residual aromatic amino groups in Y-CDs (Y-CDs-Cu2+) contributed to a newly emitted peak at 580 nm, thereby inducing fluorescence resonance energy transfer from B-CDs to Y-CDs-Cu2+. A linear correlation was found between GSH concentrations and R/B values in the range of 10-100 µM, with a limit of detection observed at 4.8 µM. By utilizing this portable device in combination with RGB analysis, the quantitative detection of GSH can be achieved even in complex food matrices such as tomatoes and grapes. The universality of this all-in-one device was further validated by pre-spraying CDs onto a paper strip for visual measurement of GSH. This work offers a portable, visual, and accessible approach to evaluating food safety and nutrition, thereby demonstrating significant economic value and holding profound implications for human health.

ICH M10 Bioanalytical Method Validation Guideline-1 year Later.

The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) adopted Guideline M10 entitled "Bioanalytical Method Validation and Study Sample Analysis" in May 2022. In October 2023, approximately one year after the adoption of the ICH M10 guideline, a "Hot Topic" session was held during the AAPS PharmSci 360 meeting to discuss the implementation of the guideline. The session focused on items the bioanalytical community felt were challenging to implement or ambiguous within the guideline. These topics included cross-validation, parallelism, comparative bioavailability studies, combination drug stability, endogenous analyte bioanalysis, and dilution QCs. In addition, the regulatory perspective on the guideline was presented. This report provides a summary of the Hot Topic session.

Inclusion of dilution quality control samples in quantitative LC-MS bioanalysis.

In LC-MS bioanalysis, sample dilution plays various roles, including bringing analyte concentrations within the validated/qualified dynamic range or alleviating matrix effect for accurate determination of the target analyte(s) in the intended study samples. Adherence to health authority requirements, incorporating good dilution practices, and timely demonstration of dilution integrity whenever samples are diluted in an analytical run are essential to ensure the reliability of bioanalytical results.

Advances and challenges in liquid chromatography-spectrometry (LC-MS) methodology for quantifying androgens and estrogens in human serum and plasma.

Accurate quantification of androgens and estrogens is critical for elucidating their roles in endocrine disorders and advancing research on their functions in human biology and pathophysiology. This review highlights recent advances and ongoing challenges in liquid chromatography- mass spectrometry (LC- MS) methodology for quantifying androgens and estrogens in human serum and plasma. We summarized current approaches for analyzing the different forms of androgens and estrogens, along with their reported levels in publications from 2010 to the present. These published levels pointed out the inconsistencies in reference intervals across studies. To address these issues, advances in derivatization methods and chromatographic separation techniques are reviewed. Future perspectives for improving the accuracy and consistency of hormone quantification in clinical and research settings were also proposed.

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.

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.

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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.

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.

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.

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.

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.

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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.

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.

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.

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.

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.