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Abstract Nowadays, there is a growing interest in innovative and more efficient therapeutics—biopharmaceuticals, based on peptides or proteins. There are increased demands on quality control of such therapeutics. One of the methods usually used for characterization and quantification of biopharmaceuticals is amino acid analysis. In this work, a modern advanced analytical method based on precolumn derivatization and reversed-phase ultra high-performance liquid chromatography in combination with single quadrupole mass spectrometer was developed for amino acid analysis in different protein samples—model sample of bovine serum albumin, sample of strong immunogenic protein keyhole limpet hemocyanin, and sample of drug etanercept present in commercially available biopharmaceutical Enbrel. The method used isotopically labeled internal standards and was validated according to the International Council for Harmonisation guideline. The developed method was characterized by favorable performance and validation parameters, such as time of analysis (6 min), specificity, linearity (r2 ≥ 0.99), limit of detection (0.009–0.822 µM), limit of quantification (1–2.5 µM), accuracy (recovery in the range 90–102.8%), intra-day (RSD in the range 0.25–11.97%) and inter-day precision (RSD in the range 1.67–11.57%), or stability (RE ≤ 12%). According to these findings, the developed amino acid analysis approach is suitable for routine use in areas of peptide/protein quantification, such as quality control laboratories of biopharmaceutical companies.

A simple analytical approach based on capillary zone electrophoresis with ultraviolet detection and repeated sample injection strategy (applied in a hydrodynamically closed separation system for the first time) was developed for the determination of ibuprofen (IBU) in commercially available pharmaceutical preparations. The proposed method was characterized by significantly increased sample throughput and favorable validation parameters, highly demanded in routine quality control laboratories. The limit of detection was predicted at the concentration level of 0.31 µg/mL. Intra-day precision expressed as the relative standard deviation of IBU concentration ranged from 1.9 to 5.6%, and corresponding intra-day accuracy expressed as the relative error was in the interval of 87.1–106.5%. Inter-day precision was in the range of 2.6–15.0%, and inter-day accuracy was 94.9–102.7%. The developed method was able to quantify IBU in complex pharmaceutical matrices represented by commercially available tablets and oral suspension. The determined contents of IBU in the tested dosage forms were in good agreement with the manufacturer’s declaration. The analytical performance of the developed method was evaluated according to the innovative RGB Additive Color Model strategy. It was demonstrated that the proposed method is characterized by very good analytical performance parameters, safety and eco-friendliness, and practical effectiveness.

Neurotransmitters (NT) are widely distributed in the central nervous system. These molecules are important for many physiological processes and the function of the immune system. Imbalance of NT are linked to numerous neurological disorders and diseases, including tauopathies. Here, a targeted approach based on on-line combination of ultra-high performance liquid chromatography with tandem mass spectrometry was validated and applied to the quantitative analysis of nine NT (acetylcholine, choline, aspartic acid, asparagine, glutamic acid, glutamine, pyroglutamate, γ-aminobutyric acid, N-acetyl-L-aspartic acid), tryptophan and its metabolite kynurenine in brain tissue samples of a rat model for tauopathy. The applied analytical method was characterized by excellent validation parameters for all analytes, such as limits of detection in the range of 0.01–1.70 µg/mL, regression coefficients of the calibration curves ≥ 0.9946, intra-day and inter-day precision expressed as coefficient of variation in the range of 0.6–11.9% and 0.6–14.4%, and accuracy in the range of 87.6–107.1% and 87.2–119.6%. Our analytical approach led to the identification of increased levels of choline and γ-aminobutyric acid in pons, and elevated concentration levels of pyroglutamate in medulla oblongata. These findings indicate that NT could play a valuable role in the study and clarification of neuroinflammation and neurodegenerative diseases.

Urine represents a convenient biofluid for metabolomic studies due to its noninvasive collection and richness in metabolites. Here, amino acids are valuable biomarkers for their ability to reflect imbalances of different biochemical pathways. An impact of amino acids on pathology, prognosis and therapy of various diseases, including inflammatory bowel disease (IBD), is therefore the subject of current clinical research. This work is aimed to develop a capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the quantification of the 20 proteinogenic amino acids in human urine samples obtained from patients suffering from IBD and treated with thiopurines. The optimized CE-MS/MS method, with minimum sample preparation (just “dilute and shoot”), exhibited excellent linearity for all the analytes (coefficients of determination were higher than 0.99), with inter-day and intra-day precision yielding relative standard deviations in the range of 0.91−15.12% and with accuracy yielding relative errors in the range of 85.47−112.46%. Total analysis time, an important parameter for the sample throughput demanded in routine practice, was shorter in ca. 17% when compared to established CE-MS methods. Favorable performance of the proposed CE-MS/MS method was also confirmed by the comparison with corresponding ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. Consistent data for the investigated amino acid metabolome were obtained using both methods. For the first time, the amino acid profiling by CE-MS approach was applied on the clinical IBD samples. Here, significant differences observed in the concentration levels of some amino acids between IBD patients undergoing thiopurine treatment and healthy volunteers could result from the simultaneous action of the disease and the corresponding therapy. These findings indicate that amino acids analysis could be a valuable tool for the study of mechanism of the IBD treatment by thiopurines.

Abstract Background Accumulation of tau leads to neuroinflammation and neuronal cell death in tauopathies, including Alzheimer’s disease. As the disease progresses, there is a decline in brain energy metabolism. However, the role of tau protein in regulating lipid metabolism remains less characterized and poorly understood. Methods We used a transgenic rat model for tauopathy to reveal metabolic alterations induced by neurofibrillary pathology. Transgenic rats express a tau fragment truncated at the N- and C-terminals. For phenotypic profiling, we performed targeted metabolomic and lipidomic analysis of brain tissue, CSF, and plasma, based on the LC-MS platform. To monitor disease progression, we employed samples from transgenic and control rats aged 4, 6, 8, 10, 12, and 14 months. To study neuron-glia interplay in lipidome changes induced by pathological tau we used well well-established multicomponent cell model system. Univariate and multivariate statistical approaches were used for data evaluation. Results We showed that tau has an important role in the deregulation of lipid metabolism. In the lipidomic study, pathological tau was associated with higher production of lipids participating in protein fibrillization, membrane reorganization, and inflammation. Interestingly, significant changes have been found in the early stages of tauopathy before the formation of high-molecular-weight tau aggregates and neurofibrillary pathology. Increased secretion of pathological tau protein in vivo and in vitro induced upregulated production of phospholipids and sphingolipids and accumulation of lipid droplets in microglia. We also found that this process depended on the amount of extracellular tau. During the later stages of tauopathy, we found a connection between the transition of tau into an insoluble fraction and changes in brain metabolism. Conclusion Our results revealed that lipid metabolism is significantly affected during different stages of tau pathology. Thus, our results demonstrate that the dysregulation of lipid composition by pathological tau disrupts the microenvironment, further contributing to the propagation of pathology.

Ostarine is frequently misused as a selective androgen receptor modulator (SARM) in sports. Consequently, there is a pressing need for reliable and simple approaches to monitor its presence in biological systems. In this work, we developed a two-dimensional analytical method utilizing online solid-phase extraction (online-SPE) in conjunction with ultra-high-performance liquid chromatography and tandem mass spectrometry (triple quadrupole). This automated 2D separation approach is characterized by minimum manual steps in the sample preparation (only dilute-and-shoot), reflecting high sample throughput and the reliability of analytical data. It provides favorable performance parameters, including a limit of detection of 0.5 pg/mL, high accuracy (relative error = 1.6–7.5%), precision (relative standard deviation = 0.8–4.5%), and sensitivity. Additionally, it demonstrates excellent linearity (r2 = 0.9999) in the calibration range of 0.05 to 25 ng/mL and robustness, with no carryover effects observed. This comparative study revealed a two-decadic-order-lower LOD of the SPE-UHPLC-MS/MS method to the corresponding UHPLC-MS/MS method and the lowest one in the group of currently published LC-MS methods. The World Anti-Doping Agency screening and confirmation criteria were met through the analysis of spiked urine samples from ten healthy volunteers. Accordingly, the proposed method is suitable for routine use in antidoping laboratories.

Clenbuterol is one of the most misused anabolic agents in professional sports. Therefore, the monitoring of clenbuterol in body fluids such as human urine is related to the development of rapid, selective and sensitive analytical methods that produce reliable results. In this work, these requirements were met by a two-dimensional separation method based on online solid-phase extraction coupled with ultra-high performance liquid chromatography–tandem mass spectrometry (SPE–UHPLC–MS/MS). The developed method provides favorable performance parameters, and it is characterized by minimum manual steps (only dilution and the addition of an internal standard) in the sample preparation. A limit of quantification (LOQ) of 0.1 ng/mL, excellent linearity (0.9999), remarkable precision (1.26% to 8.99%) and high accuracy (93.1% to 98.7%) were achieved. From a practical point of view, the analytical performance of the validated SPE–UHPLC–MS/MS method was demonstrated on blinded spiked urine samples from ten healthy volunteers. The estimated concentrations of clenbuterol were in accordance with their corresponding nominal values, as supported by the precision and accuracy data (relative standard deviation ≤5.4%, relative error ≤11%). The fulfillment of the World Anti-Doping Agency’s screening and confirmation criteria indicates that the proposed method is suitable for implementation in routine use in toxicologic and antidoping laboratories. Due to its high orthogonality and separation efficiency, the SPE–UHPLC–MS/MS method should also be easily adapted to the separation of structurally related compounds (such as clenbuterol metabolites). Thus, future antidoping applications could also include monitoring of clenbuterol metabolites, providing a longer detection widow.

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized—GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.

A capillary electrophoresis–tandem mass spectrometry method with a multisegment injection and an in-capillary field-enhanced sample stacking for determination of therapeutic peptide triptorelin in pharmaceutical and biological matrices was developed. The CE separation conditions were optimized in order to obtain maximal separation efficiency, analytical signal intensity and stability, and minimal adsorption of the analyzed peptide onto the capillary wall (1 M formic acid—HFo, pH 1.88). The implementation of the field-enhanced sample injection into CE improved the value of limit of detection 50 times while the multisegment injection increased the sample throughput three times in comparison to a conventional CE approach. The proposed method was characterized by favorable performance parameters, such as linearity (r2 ≥ 0.99), limit of detection (5 ng mL−1 in water matrix, 25 ng mL−1 in plasma matrix), precision (relative standard deviation, 1.5–9.4% for intraday and 2.3–11.9% for interday reproducibility), or accuracy (relative errors in the range of 80–109%). The FDA-validated method was successfully applied to the analysis of triptorelin in the commercial drug Diphereline® 0.1 mg (powder for injection) and in spiked human plasma samples. Favorable performance parameters along with proven application potentialities indicate the usefulness of the proposed method for its routine use in drug quality control laboratories and for clinical analysis, such as determination of triptorelin levels in plasma (for pharmacokinetic study).

Oxandrolone, a synthetic testosterone analog, is used for the treatment of several diseases associated with weight loss. Unfortunately, oxandrolone is abused by many athletes and bodybuilders due to its strong anabolic effect. We have developed and validated a highly sensitive and rapid on-line SPE-UHPLC-MS/MS method for the determination of oxandrolone and simultaneous identification of its major metabolite 17-epi-oxandrolone in urine matrices. Enrichment of the analytes via an integrated solid-phase extraction was achieved using an Acquity UPLC BEH C18 Column. Subsequently, the chromatographic separation of the on-line preconcentrated sample fraction was achieved using an Acquity HSS T3 C18 Column. For the structural identification of these analytes, a high-resolution mass spectrometer Synapt-G2Si coupled to the Acquity M-class nano-LC system with ionKey source was used. A highly sensitive determination of oxandrolone was achieved using a tandem quadrupole mass spectrometer XEVO TQD. The method was successfully validated in the linear range of oxandrolone from 81.63 pg·mL−1 (limit of quantification, LOQ) to 5000 pg·mL−1 in the human urine matrix. It was applied to the analysis of real urine samples obtained from a healthy volunteer after the oral administration of one dose (10 mg) of oxandrolone. Concentration vs. time dependence was tested in the time interval of 4 h–12 days (after oral administration) to demonstrate the ability of the method to detect the renal elimination of oxandrolone from the human body. Favorable performance parameters along with successful application indicate the usefulness of the proposed method for its routine use in antidoping control labs.

Azathioprine is the main thiopurine drug used in the treatment of immune-based inflammations of gastrointestinal tract. For the purpose of therapy control and optimization, effective and reliable analytical methods for a rapid drug monitoring in biological fluids are essential. Here, we developed a separation method based on the capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) for the simultaneous determination of azathioprine and its selected metabolites (6-thioguanine, 6-mercaptopurine, and 6-methylmercaptopurine) as well as other co-medicated drugs (mesalazine, prednisone, and allopurinol). The optimized CE-MS/MS conditions provided a very efficient and stable system for the separation and sensitive detection of these drugs in human urine matrices. The developed method was successfully applied for the assay of the targeted drugs and their selected metabolites in urine samples collected from patients suffering from inflammatory bowel disease (IBD) and receiving azathioprine therapy. The developed CE-MS/MS method, due to its reliability, short analysis time, production of complex clinical profiles, and favorable performance parameters, evaluated according to FDA guidelines for bioanalytical method validation, is proposed for routine clinical laboratories to optimize thiopurine therapy, estimate enzymatic activity, and control patient compliance with medication and co-medication.

An advanced two-dimensional capillary electrophoresis method, based on on-line combination of capillary isotachophoresis and capillary zone electrophoresis with cyclodextrin additive in background electrolyte, was developed for effective determination of serotonin in human urine. Hydrodynamically closed separation system and large bore capillaries (300–800 µm) were chosen for the possibility to enhance the sample load capacity, and, by that, to decrease limit of detection. Isotachophoresis served for the sample preseparation, defined elimination of sample matrix constituents (sample clean up), and preconcentration of the analyte. Cyclodextrin separation environment enhanced separation selectivity of capillary zone electrophoresis. In this way, serotonin could be successfully separated from the rest of the sample matrix constituents migrating in capillary zone electrophoresis step so that human urine could be directly (i.e., without any external sample preparation) injected into the analyzer. The proposed method was successfully validated, showing favorable parameters of sensitivity (limit of detection for serotonin was 2.32 ng·mL−1), linearity (regression coefficient higher than 0.99), precision (repeatability of the migration time and peak area were in the range of 0.02–1.17% and 5.25–7.88%, respectively), and recovery (ranging in the interval of 90.0–93.6%). The developed method was applied for the assay of the human urine samples obtained from healthy volunteers. The determined concentrations of serotonin in such samples were in the range of 12.4–491.2 ng·mL−1 that was in good agreement with literature data. This advanced method represents a highly effective, reliable, and low-cost alternative for the routine determination of serotonin as a biomarker in human urine.

Nowadays, lipidomics plays a crucial role in the investigation of novel biomarkers of various diseases. Its implementation into the field of clinical analysis led to the identification of specific lipids and/or significant changes in their plasma levels in patients suffering from cancer, Alzheimer’s disease, sepsis, and many other diseases and pathological conditions. Profiling of lipids and determination of their plasma concentrations could also be helpful in the case of drug therapy management, especially in combination with therapeutic drug monitoring (TDM). Here, for the first time, a combined approach based on the TDM of colistin, a last-resort antibiotic, and lipidomic profiling is presented in a case study of a critically ill male patient suffering from Pseudomonas aeruginosa-induced pneumonia. Implementation of innovative analytical approaches for TDM (online combination of capillary electrophoresis with tandem mass spectrometry, CZE-MS/MS) and lipidomics (liquid chromatography–tandem mass spectrometry, LC-MS/MS) was demonstrated. The CZE-MS/MS strategy confirmed the chosen colistin drug dosing regimen, leading to stable colistin concentrations in plasma samples. The determined colistin concentrations in plasma samples reached the required minimal inhibitory concentration of 1 μg/mL. The complex lipidomics approach led to monitoring 545 lipids in collected patient plasma samples during and after the therapy. Some changes in specific individual lipids were in good agreement with previous lipidomics studies dealing with sepsis. The presented case study represents a good starting point for identifying particular individual lipids that could correlate with antimicrobial and inflammation therapeutic management.

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis – tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis – mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis.

Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4′-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5–8.2% and 2.3–7.4%, accuracy was in the range of 93.3–109.7% and 94.7–110.1%, limits of detection (LODs) were in the range of 0.15–9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer’s disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

Monitoring plasma concentrations of β-lactam antibiotics is crucial, particularly in critically ill patients, where variations in concentrations can lead to treatment failure or adverse events. Standardized antimicrobial regimens may not be effective for all patients, especially in special groups with altered physiological parameters. Pharmacokinetic/pharmacodynamic (PK/PD) studies highlight the time-dependent antibacterial activity of these antibiotics, emphasizing the need for personalized dosing. Therapeutic drug monitoring (TDM) is essential, requiring rapid and accurate analytical methods for precise determination of drugs in biological material (typically plasma or serum). This study presents a novel capillary zone electrophoresis–tandem mass spectrometry (CZE-MS/MS) method designed for the simultaneous quantification of five penicillin antibiotics, two cephalosporins, one carbapenem, and two β-lactamase inhibitors in a single run. The method involves a simple sample pretreatment—precipitation with organic solvent—and has a run time of 20 min. Optimization of CZE separation conditions revealed that 20 mM ammonium hydrogen carbonate (NH4HCO3) serves as the optimal background electrolyte (BGE). Positive electrospray ionization (ESI) mode, with isopropyl alcohol (IP)/10 mM ammonium formate water solution (50/50, v/v) as the sheath liquid, was identified as the optimal condition for MS detection. Method validation according to the Food and Drug Administration (FDA) guideline for development of bioanalytical methods demonstrated satisfactory selectivity, linearity, recovery, robustness, and stability. The method’s practicality was evaluated using the Blue Applicability Grade Index (BAGI), yielding a score of 77.5. Moreover, the greenness of the proposed method was evaluated by two commonly used metric tools—Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI). The developed CZE-MS/MS method offers a practical and reliable approach for quantifying a broad spectrum of β-lactam antibiotics in plasma. Its ability to simultaneously quantify multiple analytes in a single run, coupled with a straightforward sample pretreatment, positions it as a valuable and prospective tool for TDM in critically ill patients.

Short-chain fatty acids (SCFAs) are the main metabolites produced by bacterial fermentation of non-digestible carbohydrates in the gastrointestinal tract. They can be seen as the major flow of carbon from the diet, through the microbiome to the host. SCFAs have been reported as important molecules responsible for the regulation of intestinal homeostasis. Moreover, these molecules have a significant impact on the immune system and are able to affect inflammation, cardiovascular diseases, diabetes type II, or oncological diseases. For this purpose, SCFAs could be used as putative biomarkers of various diseases, including cancer. A potential diagnostic value may be offered by analyzing SCFAs with the use of advanced analytical approaches such as gas chromatography (GC), liquid chromatography (LC), or capillary electrophoresis (CE) coupled with mass spectrometry (MS). The presented review summarizes the importance of analyzing SCFAs from clinical and analytical perspective. Current advances in the analysis of SCFAs focused on sample pretreatment, separation strategy, and detection methods are highlighted. Additionally, it also shows potential areas for the development of future diagnostic tools in oncology and other varieties of diseases based on targeted metabolite profiling.

Lipids represent a large group of biomolecules that are responsible for various functions in organisms. Diseases such as diabetes, chronic inflammation, neurological disorders, or neurodegenerative and cardiovascular diseases can be caused by lipid imbalance. Due to the different stereochemical properties and composition of fatty acyl groups of molecules in most lipid classes, quantification of lipids and development of lipidomic analytical techniques are problematic. Identification of different lipid species from complex matrices is difficult, and therefore individual analytical steps, which include extraction, separation, and detection of lipids, must be chosen properly. This review critically documents recent strategies for lipid analysis from sample pretreatment to instrumental analysis and data interpretation published in the last five years (2019 to 2023). The advantages and disadvantages of various extraction methods are covered. The instrumental analysis step comprises methods for lipid identification and quantification. Mass spectrometry (MS) is the most used technique in lipid analysis, which can be performed by direct infusion MS approach or in combination with suitable separation techniques such as liquid chromatography or gas chromatography. Special attention is also given to the correct evaluation and interpretation of the data obtained from the lipid analyses. Only accurate, precise, robust and reliable analytical strategies are able to bring complex and useful lipidomic information, which may contribute to clarification of some diseases at the molecular level, and may be used as putative biomarkers and/or therapeutic targets.

Kynurenines have immunomodulatory and neuroactive properties and can influence the central nervous system. Previous studies showed the involvement of the kynurenines in the pathogenesis and progression of neurodegenerative disease. In neurodegenerative disorders, including tauopathies, the tryptophan metabolism is shifted toward neurotoxic agents and the reduction of neuroprotectant products. Astrocyte-derived kynurenic acid serves as a neuroprotectant. However, systemic administration of kynurenic acid is not effective because of low permeability across the blood-brain barrier (BBB). We used a kynurenic acid analog with similar biological activity but higher brain permeability to overcome BBB limitations. In the present study, we used amide derivate of kynurenic acid N-(2-N, N-dimethylaminoethyl)− 4-oxo-1 H-quinoline-2-carboxamid (KYNA-1). We administered KYNA-1 for three months to tau transgenic rats SHR-24 and analyzed the effect on tau pathology and activation of glial cells. Primary glial cell cultures were applied to identify the mechanism of the KYNA-1 effect. KYNA-1 was not toxic to rats after chronic three-month administration. When chronically administered, KYNA-1 reduced hyperphosphorylation of insoluble tau in the brain of transgenic rats. Noteworthily, the plasma total tau was also reduced. We determined that the effect of KYNA-1 on tau pathology was induced through the modulation of glial activation. KYNA-1 inhibited LPS induced activation of astrocytes and induced transformation of microglia to M2 phenotype. We identified that the administration of KYNA-1 reduced tau hyperphosphorylation and neuroinflammation. KYNA-1 may serve as a promising treatment for tauopathies.

Summary: Background: The emergence of new SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) that harbor mutations in the viral S protein raised concern about activity of current vaccines and therapeutic antibodies. Independent studies have shown that mutant variants are partially or completely resistant against some of the therapeutic antibodies authorized for emergency use. Methods: We employed hybridoma technology, ELISA-based and cell-based S-ACE2 interaction assays combined with authentic virus neutralization assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize the new variants of SARS-CoV-2. Findings: AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). In addition, AX677 is able to bind Omicron Spike protein just like the wild type Spike. The combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. Prophylactic administration of AX290 and AX677, either individually or in combination, effectively reduced viral burden and inflammation in the lungs, and prevented disease in a mouse model of SARS-CoV-2 infection. Interpretation: The virus-neutralizing properties were fully reproduced in chimeric mouse-human versions of the antibodies, which may represent a promising tool for COVID-19 therapy. Funding: The study was funded by AXON Neuroscience SE and AXON COVIDAX a.s.

In the presented study, a capillary electrophoresis-mass spectrometry method combining high separation efficiency and sensitive detection has been developed and validated, for the first time, to quantify branched chain amino acids (valine, isoleucine, leucine) in commercial food and sport supplement samples and human plasma samples. The separations were performed in a bare fused silica capillary. The background electrolyte was composed of 500 mM formic acid with pH 2.0. The plasma sample pretreatment was realized by simple protein precipitation with acetonitrile. Injection of a short zone of highly basic electrolyte before the sample injection and application of the negative pressure on the separation were accompanied by enhanced resolution of the isobaric amino acids—isoleucine and leucine. The developed method was characterized by favorable validation parameters, such as linearity (r2 > 0.99), accuracy and precision, the limit of detection, lower limit of quantification, or robustness. These parameters were more than sufficient for the quantification of branched chain amino acids in various samples. The determined concentrations of branched chain amino acids in food and sports supplements were in very good agreement with the content declared by the manufacturer. The investigated concentrations of branched chain amino acids were in the range 294.68–359.24 µM for valine, 91.76–95.67 µM for isoleucine, and 196.78–251.24 µM for leucine. These concentrations fall within the physiological limits. The developed CE-MS/MS method represents a suitable alternative to traditional approaches used in branched chain amino acid quality control and bioanalysis.

Autism spectrum disorder is a heterogeneous neurodevelopmental disease. Currently, no biomarker of this disease is known. Diagnosis is performed through observation, standardized behavioral scales, and interviews with parents. In practice, diagnosis is often delayed to the average age of four years or even more which adversely affects a child’s perspective. A laboratory method allowing to detect the disorder at earlier stages is of a great need, as this could help the patients to start with treatment at a younger age, even prior to the clinical diagnosis. Recent evidence indicates that metabolomic markers should be considered as diagnostic markers, also serving for further differentiation and characterization of different subgroups of the autism spectrum. In this study, we developed an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry method for the simultaneous determination of six metabolites in human urine. These metabolites, namely methylguanidine, N-acetyl arginine, inosine, indole-3-acetic acid, indoxyl sulfate and xanthurenic acid were selected as potential biomarkers according to prior metabolomic studies. The analysis was carried out by means of reversed-phase liquid chromatography with gradient elution. Separation of the metabolites was performed on a Phenomenex Luna® Omega Polar C18 (100 × 1.0 mm, 1.6 µm) column at a flow rate of 0.15 mL/min with acetonitrile/water 0.1% formic acid aqueous as the mobile phase. The analysis was performed on a group of children with autism spectrum disorder and age-matched controls. In school children, we have detected disturbances in the levels of oxidative stress markers connected to arginine and purine metabolism, namely methylguanidine and N-acetylargine. Also, products of gut bacteria metabolism, namely indoxyl sulfate and indole-3-acetic acid, were found to be elevated in the patients’ group. We can conclude that this newly developed method is fast, sensitive, reliable, and well suited for the quantification of proposed markers.