Implementation of Modern Therapeutic Drug Monitoring and Lipidomics Approaches in Clinical Practice: A Case Study with Colistin Treatment.

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.

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 and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples.

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.

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.

Amino acids in inflammatory bowel diseases: Modern diagnostic tools and methodologies.

Amino acids are crucial building blocks of living organisms. Together with their derivatives, they participate in many intracellular processes to act as hormones, neuromodulators, and neurotransmitters. For several decades amino acids have been studied for their potential as markers of various diseases, including inflammatory bowel diseases. Subsequent improvements in sample pretreatment, separation, and detection methods have enabled the specific and very sensitive determination of these molecules in multicomponent matrices-biological fluids and tissues. The information obtained from targeted amino acid analysis (biomarker-based analytical strategy) can be further used for early diagnostics, to monitor the course of the disease or compliance of the patients. This review will provide an insight into current knowledge about inflammatory bowel diseases, the role of proteinogenic amino acids in intestinal inflammation and modern analytical techniques used in its diagnosis and disease activity monitoring. Current advances in the analysis of amino acids focused on sample pretreatment, separation strategy, or detection methods are highlighted, and their potential in clinical laboratories is discussed. In addition, the latest clinical data obtained from the metabolomic profiling of patients suffering from inflammatory bowel diseases are summarized with a focus on proteinogenic amino acids.

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).

Capillary Electrophoresis-Mass Spectrometry with Multisegment Injection and In-Capillary Preconcentration for High-Throughput and Sensitive Determination of Therapeutic Decapeptide Triptorelin in Pharmaceutical and Biological Matrices.

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).

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.

Ultrasensitive determination of serotonin in human urine by a two dimensional capillary isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry.

A two-dimensional capillary isotachophoresis-capillary zone electrophoresis method hyphenated with tandem mass spectrometry was developed and validated for ultrasensitive quantification of serotonin in real human urine samples. Under optimal conditions, the separation was achieved within 12 min (including on-line sample preparation) with the limit of detection of 34 pg mL-1 (due to a large volume sample injection, here 10 µL, and isotachophoretic preconcentration). This concentration limit represents the lowest value for serotonin in comparison to other previously published separation methods employing mass spectrometry detection and applied to urine matrices. Thanks to high orthogonality, on-line concentration and clean-up effects of this approach, other excellent validation parameters such as linearity (coefficient of determination > 0.99), inter-day and intra-day precision (relative standard deviations 3.5-12.2%), accuracy (relative errors within 99-109.4%), and recovery (96-102%) could be easily obtained too. To demonstrate applicability of the method, we monitored serotonin levels in various real samples (from a healthy volunteer and clinical ones). The determined levels, normalized on the creatinine concentrations, were in the range of 6.81-12.86 ng mmol-1 creatinine This advanced method is suggested for an effective, reliable, high sample throughput, and low cost routine clinical screening or targeted metabolomic studies of serotonin in urine samples.

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.