Changes in lipid metabolism track with the progression of neurofibrillary pathology in tauopathies.

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.

Quantitative analysis of therapeutic peptides by CZE using multiple sample injection in hydrodynamically closed separation system.

Therapeutic peptides have emerged as an innovative and promising class of therapeutic compounds in modern medicine. Synthetic peptide analogs triptorelin and lanreotide are known for their pronounced clinical versatility and potency. In this study, we present the development and validation of novel methods based on capillary zone electrophoresis performed in hydrodynamically closed system (HCS) and paired with ultraviolet detection and repeated injection sample introduction. To the best of our knowledge, we developed the first capillary electrophoresis-based method for the determination of lanreotide, and concurrently, the first HCS method for the determination of triptorelin. Maximal separation efficiency and signal intensity were achieved using background electrolytes composed of 50 mM formic acid with the addition of 0.05% (v/v) methyl-hydroxyethyl cellulose. The proposed methods exhibit favorable performance characteristics, namely, calibration curve (r2 exceeding 0.99), low limits of detection (0.25 µg/mL in a water matrix and 0.5 µg/mL in synthetic urine), acceptable precision (relative standard deviation ranging from 2.2% to 9.6% for intraday repeatability and between 5.2% and 14.9% for interday reproducibility), and accuracy (relative errors falling within the 91.1%-107.8% range). The method for triptorelin determination was then used for its quantification in a commercially available drug dosage form (powder for injection) and in spiked synthetic urine samples. The developed methods were also evaluated according to the novel blue applicability grade index, revealing their superior applicability. The results collectively point out the potential of the proposed methods for both quality control and clinical investigations.

Capillary electrophoresis in the analysis of therapeutic peptides-A review.

Therapeutic peptides are a growing class of innovative drugs with high efficiency and a low risk of adverse effects. These biomolecules fall within the molecular mass range between that of small molecules and proteins. However, their inherent instability and potential for degradation underscore the importance of reliable and effective analytical methods for pharmaceutical quality control, therapeutic drug monitoring, and compliance testing. Liquid chromatography-mass spectrometry (LC-MS) has long time been the "gold standard" conventional method for peptide analysis, but capillary electrophoresis (CE) is increasingly being recognized as a complementary and, in some cases, superior, highly efficient, green, and cost-effective alternative technique. CE can separate peptides composed of different amino acids owing to differences in their net charge and size, determining their migration behavior in an electric field. This review provides a comprehensive overview of therapeutic peptides that have been used in the clinical environment for the last 25 years. It describes the properties, classification, current trends in development, and clinical use of therapeutic peptides. From the analytical point of view, it discusses the challenges associated with the analysis of therapeutic peptides in pharmaceutical and biological matrices, as well as the evaluation of CE as a whole and the comparison with LC methods. The article also highlights the use of microchip electrophoresis, nonaqueous CE, and nonconventional hydrodynamically closed CE systems and their applications. Overall, the article emphasizes the importance of developing new CE-based analytical methods to ensure the high quality, safety, and efficacy of therapeutic peptides in clinical practice.

Recent Analytical Methodologies in Lipid Analysis.

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.

Comparison of 1D a 2D ITP-MS performance parameters and application possibilities: Ultratrace determination of B vitamins in human urine.

The possibility to investigate analytes at ultra-low concentration levels still remains a hot topic in bioanalysis. In this area, various preconcentration techniques are an integral part of analytical procedures. When applying electromigration separation techniques, an isotachophoresis has been advantageously employed many times for this purpose. To solve current biomedical tasks effectively, an advanced two-dimensional isotachophoretic instrument (in a hydrodynamically closed separation system with an enhanced sample load capacity) hyphenated with mass spectrometry (ITP-ITP-MS) has been proposed by Foret and coworkers. As a continuation, this work represents the first study dealing with a full validation of an ITP-ITP-MS method. In order to see the benefits of an online ITP sample pretreatment (preconcentration and clean-up) on the performance parameters, the developed 2D ITP-MS method was compared with a corresponding 1D ITP-MS method. Application potentialities of the compared methods were demonstrated via a determination of two B vitamins, namely thiamine and pyridoxine, in human urine samples. The developed 2D ITP-MS method showed its enhanced effectivity and usefulness for a routine biomedical use (here, a reliable screening of trace B vitamins in human urine without an offline sample preparation).

An Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Determination of 4 ß-Lactam Antibiotics, Tazobactam, and Linezolid in Human Plasma Samples.

BACKGROUND: Optimization of antimicrobial therapy is a challenge in critically ill patients who develop extreme interindividual and intraindividual pharmacokinetic variability. Therapeutic drug monitoring is a valuable tool for maximizing the effect of a drug and minimizing its adverse and unwanted effects. The aim of the current work was to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine multiple antibiotics in clinical plasma samples from critically ill patients; low sample volume and rapid processing of samples were considered the main criteria. METHODS: A separation method based on an online combination of UHPLC-MS/MS was developed for the simultaneous determination of 4 ß-lactam antibiotics (cefepime, meropenem, cefotaxime, and piperacillin), tazobactam, and linezolid in human plasma samples. The volume of plasma sample used for analysis was 20 µL. The developed method was validated according to Food and Drug Administration guidelines. RESULTS: The chromatographic run time was 8 minutes. Calibration curves were linear for concentration ranges of 0.1-100 mcg/mL (r 2 > 0.99) for tazobactam, meropenem, cefotaxime, linezolid, and piperacillin and 1-100 mcg/mL (r 2 > 0.99) for cefepime. The intraday and interday accuracy of the method ranged from 92.4% to 110.7% and 93.6% to 113.3%, respectively. The intraday and interday precision values were ≤17.3% and ≤17.4%, respectively. No interfering and carryover analytes were observed. CONCLUSIONS: The developed UHPLC-MS/MS method is an appropriate and practical tool for therapeutic drug monitoring of the selected antibiotics. Owing to its rapidity, requirement of low sample volume, and high selectivity, sensitivity, and reliability, it can be effectively implemented in routine clinical laboratory tests for critically ill patients.

Capillary zone electrophoresis in combination with UV detection for simultaneous determination of tramadol and paracetamol in pharmaceutical and biological samples.

The aim of the present study is the development and validation of a simple method based on capillary zone electrophoresis coupled with UV detection for simultaneous determination of tramadol and paracetamol in pharmaceutical and biological samples. The background electrolyte was composed of 50 mM ammonium carbonate, which is a type of a non-conventional electrolyte system. The developed method was characterized by suitable validation parameters, such as linearity (coefficient of determination r2 0,995), selectivity or the limit of detection at the level of 0.25 - 0.5 μg/ml. Acceptable values of accuracy and precision were obtained, which were in good agreement with the recommended validation guidelines for analysis of pharmaceutical and biological samples. Detection was performed at a wavelength of 200 nm. The developed method was successfully applied to determine tramadol and paracetamol in various dosage forms and in urine biological samples. Achieved results indicate a potential of the method to be integrated in the common quality control processes of drugs and/or in bioanalysis.

Determination of Branched-Chain Amino Acids in Food Supplements and Human Plasma by a CE-MS/MS Method with Enhanced Resolution.

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.

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy.

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.

Fast and Sensitive Screening of Oxandrolone and Its Major Metabolite 17-Epi-Oxandrolone in Human Urine by UHPLC-MS/MS with On-Line SPE Sample Pretreatment.

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.

Capillary electrophoresis and ultra-high-performance liquid chromatography methods in clinical monitoring of creatinine in human urine: A comparative study.

Creatinine is an important diagnostic marker and is also used as a standardization tool for the quantitative evaluation of exogenous/endogenous substances in urine. This study aimed at evaluating and comparing three analytical approaches, based on hyphenations of different separation [two-dimensional capillary isotachophoresis (CITP-CITP), capillary zone electrophoresis (CZE), ultra-high-performance liquid chromatography (UHPLC)] and detection [conductivity (CD), ultraviolet (UV), tandem mass spectrometry (MS/MS)] techniques, for their ability to provide reliable clinical data along with their suitability for the routine clinical use (cost, simplicity, sample throughput). The developed UHPLC-MS/MS, CITP-CITP-CD, and CZE-UV methods were characterized by favorable performance parameters, such as linearity (r ˃ 0.99), precision (relative standard deviation, 0.22-2.97% for the creatinine position in analytical profiles), and recovery (87.1-115.1%). Clinical data, obtained from the analysis of 24 human urine samples by a reference enzymatic method, were comparable with those obtained by the tested methods (Passing-Bablok regression and Bland-Altman analysis), approving their usefulness for the routine clinical use. In this context, the UHPLC-MS/MS method provides benefits of enhanced orthogonality/accuracy and high sample throughput (threefold shorter total analysis times than the CE methods), whereas advantages of the CE methods for routine labs are simplicity and low cost of both the instrumentation and measurements.

Determination of thiamine and pyridoxine in food supplements and beverages by the simple capillary zone electrophoresis in combination with UV detection.

The paper is focused on development of a simple analytical method based on capillary zone electro-phoresis in combination with UV-detection for simul-taneous detemination of thiamine and pyridoxine in pharmaceutical and food samples. The separation of thiamine and pyridoxine was performed in a background electrolyte composed of 25 mmol l-1 GABA + 50 mmol l-1 HAc+ 0.05% m-HEC. The UV detector was set at the constant wavelength of 260 nm. Limit of detection was 0.059 µg ml-1 for thiamine and 0.23 µg ml-1 for pyridoxine. These levels suggest that relatively low quantities of thiamine and pyridoxine can be detected. The presented CZE-UV method enabled effective determination of the two vitamins in 5 food supplements and 11 energy drinks and vitamin waters.

Profiling of Amino Acids in Urine Samples of Patients Suffering from Inflammatory Bowel Disease by Capillary Electrophoresis-Mass Spectrometry.

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.

Comparison of hydrodynamically closed two-dimensional capillary electrophoresis coupled with ultraviolet detection and hydrodynamically open capillary electrophoresis hyphenated with mass spectrometry in the bioanalysis of varenicline.

Two capillary electrophoresis methods for monitoring renally excreted varenicline, a highly effective drug prescribed for smoking cessation, in human urine were developed and compared. A method combining capillary electrophoresis with mass spectrometry was proposed for the fast analysis of varenicline (analysis time up to 7 min). Here, mass spectrometry was a prerequisite for achieving high sensitivity and selectivity of the analysis suitable for the quantification of a 15 ng/mL level of varenicline in un-pretreated urine matrices. An alternative approach, two-dimensional (column-coupled) capillary electrophoresis with enhanced sample load capacity and ultraviolet detection, was proposed as a low-cost alternative to capillary electrophoresis with mass spectrometry. The isotachophoresis on-line sample treatment included simple elimination of the major matrix constituents and stacking of the sample in a large volume so that threefold lower quantitation limits could be easily achieved in comparison to the capillary electrophoresis with mass spectrometry. On the other hand, longer analysis time (ca. 4.5-fold) and more complex electrolyte system in the coupled zone electrophoresis step (including two additives enhancing separation selectivity, i.e. isopropanol and cyclodextrin) were prerequisites for the complete separation of varenicline from the sample matrix. Anyway, both the developed methods were validated according to the Food and Drug Administration guidelines showing favorable performance parameters, suitable for their routine biomedical use.

Two-Dimensional Capillary Electrophoresis with On-Line Sample Preparation and Cyclodextrin Separation Environment for Direct Determination of Serotonin in Human Urine.

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.

Capillary Electrophoresis Hyphenated with Mass Spectrometry for Determination of Inflammatory Bowel Disease Drugs in Clinical Urine Samples.

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.

Enantioselective column coupled electrophoresis employing large bore capillaries hyphenated with tandem mass spectrometry for ultra-trace determination of chiral compounds in complex real samples.

A new multidimensional analytical approach for the ultra-trace determination of target chiral compounds in unpretreated complex real samples was developed in this work. The proposed analytical system provided high orthogonality due to on-line combination of three different methods (separation mechanisms), i.e. (1) isotachophoresis (ITP), (2) chiral capillary zone electrophoresis (chiral CZE), and (3) triple quadrupole mass spectrometry (QqQ MS). The ITP step, performed in a large bore capillary (800 µm), was utilized for the effective sample pretreatment (preconcentration and matrix clean-up) in a large injection volume (1-10 µL) enabling to obtain as low as ca. 80 pg/mL limits of detection for the target enantiomers in urine matrices. In the chiral CZE step, the different chiral selectors (neutral, ionizable, and permanently charged cyclodextrins) and buffer systems were tested in terms of enantioselectivity and influence on the MS detection response. The performance parameters of the optimized ITP - chiral CZE-QqQ MS method were evaluated according to the FDA guidance for bioanalytical method validation. Successful validation and application (enantioselective monitoring of renally eliminated pheniramine and its metabolite in human urine) highlighted great potential of this chiral approach in advanced enantioselective biomedical applications.

An Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method with Online Solid-Phase Extraction Sample Preparation for the High-Throughput and Sensitive Determination of Ostarine in Human Urine.

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.

Development and Validation of a Capillary Zone Electrophoresis-Tandem Mass Spectrometry Method for Simultaneous Quantification of Eight ß-Lactam Antibiotics and Two ß-Lactamase Inhibitors in Plasma Samples.

Monitoring plasma concentrations of ß-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 ß-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 ß-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.

A simple UHPLC-MS/MS method for determination of SET2, a selective antagonist of TRPV2 receptor, in rat plasma samples.

Targeting the transient receptor potential vanilloid 2 channels (TRPV2) in order to alleviate or reverse the course of several diseases including multiple cancers, cardiovascular, immunological, or neurological disorders have been a matter of focus for several years now. SET2, a selective TRPV2 inhibitor, represents an innovative molecule which came into recognition in 2019 and seems to be a promising therapeutic modality in cancer and cardiac diseases. Drug discovery and bioanalysis in clinical environment demands simple, excellent, highly reliable, fast, sensitive, and selective analytical approaches which enable unambiguous identification and quantification of demanded molecule. Here, a targeted ultra-high-performance liquid chromatography - tandem mass spectrometry with electrospray ionization was developed for the quantification of SET2 in plasma samples. The developed method enabled analysis of approx. 15 samples within one hour. Simplicity of the whole analytical procedure can be emphasized by a very simple sample pretreatment based only on the protein precipitation with organic acid (here, 2 M tricholoroacetic acid). The validation procedure was characterized by promising validation parameters and excellent sensitivity what was documented by the limit of detection value at pg.mL-1 concentration level. Analytical validation reported intra- and interday accuracy < 15 % for all quality control samples concentration levels. Similarly, excellent level of intra- (0.1 - 4.8 %) and interday (0.5 - 3.3 %) precision for the tested quality control samples was obtained. The applicability of the developed method was proven by quantifying SET2 concentration levels in plasma samples obtained from Wistar rats that were administered this drug intraperitoneally at a dose of 25 mg/kg. We expect that our new analytical method represents a very attractive tool that could be easily implemented in pharmacokinetics studies and/or therapeutic drug monitoring. Moreover, its applicability was confirmed by the new practicability evaluation metric tool.