Production of antibody and development of enzyme-linked immunosorbent assay for therapeutic drug monitoring of eravacycline.

Eravacycline (ERC) was approved for clinical use in 2018. It is more potent than other tetracyclines and can overcome resistance, making it an attractive option for combating multidrug-resistant bacterial infections. Intensive pharmacokinetic (PK) studies are currently being conducted to ensure the effectiveness and safety of ERC in various groups of patients, including those undergoing extracorporeal therapies. This study is the first attempt to develop a simple, efficient, and high-throughput immunoassay for quantifying ERC in human or animal serum. BSA-ERC conjugate as immunogen elicited antibody production in rabbits. Monitoring of the immune response and comparison of homologous and heterologous coating antigens allowed selection of immunoreagents and development of an assay that was selective for ERC possessing sensitivity (IC50), dynamic range (IC20-IC80) and detection limit equal to 3.3 ng/mL, 0.27-54 ng/mL and 0.09 ng/mL, respectively. The developed ELISA showed acceptable recovery of ERC (85-105 %) from rabbit and human serum in the clinically relevant concentration range of 0.1-3.0 mg/L. The method was used to quantify serum ERC concentration in the pilot PK study in Soviet chinchilla rabbits. The results were confirmed by HPLC-MS/MS.

Analysis of 4-Hydroxyphenyllactic Acid and Other Diagnostically Important Metabolites of α-Amino Acids in Human Blood Serum Using a Validated and Sensitive Ultra-High-Pressure Liquid Chromatography-Tandem Mass Spectrometry Method.

The profile of and dynamic concentration changes in tyrosine, phenylalanine, and tryptophan metabolites in blood are of great interest since they could be considered potential biomarkers of different disorders. Some aromatic metabolites, such as 4-hydroxyphenyllactic, 4-hydroxyphenylacetic, phenyllactic, and 4-hydroxybenzoic acids have previously demonstrated their diagnostic significance in critically ill patients and patients with post-COVID-19 syndrome. In this study, a sensitive method, including serum protein precipitation with methanol and ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection, was developed and validated for six phenyl- and five indole-containing acids in human serum. The liquid-liquid extraction was also examined, but it demonstrated unsatisfactory results based on analyte recoveries and the matrix effect. However, the recoveries for all analytes reached 100% and matrix effects were not observed using protein precipitation. This made it possible to use deionized water as a blank matrix. The lower limits of quantitation (LLOQs) were from 0.02 to 0.25 µmol/L. The validated method was used for the analysis of serum samples of healthy volunteers (n = 48) to reveal the reference values of the target analytes. The concentrations of the most clinically significant metabolite 4-hydroxyphenyllactic acid, which were revealed using UPLC-MS/MS and a previously developed gas chromatography-mass spectrometry method, were completely comparable. The proposed UPLC-MS/MS protocol can be used in the routine clinical practice of medical centers.

Microextraction of aromatic microbial metabolites by packed hypercrosslinked polystyrene from blood serum.

The article is devoted to the application of modern sample preparation technique - microextraction by packed sorbent (MEPS) - in conjunction with non-conventional type of sorbent - hypercrosslinked polystyrene, that was investigated for the first time in this work. Their combination was used to extract phenylcarboxylic acid-type aromatic microbial metabolites from serum samples of a healthy volunteer with following derivatization and GC-MS detection. As barrel insert and needle for MEPS with hypercrosslinked polystyrene is not produced, we designed a device to imitate the commercial MEPS system with packed granular biporous hypercrosslinked polystyrene. Nine aromatic microbial metabolites, including sepsis associated phenyllactic, 4-hydroxyphenyllactic and 4-hydroxyphenylacetic acids, were extracted from serum samples (recoveries were 20-70%) and a linear dependence was revealed in the most clinically significant range of concentrations (0.5-18 µM). The results obtained demonstrate the perspective of the applying of hypercrosslinked polystyrene in commercial devices for MEPS for the future analyses of biological samples, in particular for the early diagnosis of sepsis and treatment effectiveness control.

Analysis of phenylcarboxylic acid-type microbial metabolites by microextraction by packed sorbent from blood serum followed by GC-MS detection.

A method for analysis of 8 phenylcarboxylic acids in blood serum was developed based on the coupling of microextraction by packed sorbent, derivatization and GC-MS detection. These compounds are low molecular weight aromatic microbial metabolites that are proven and prospective indicators of sepsis in critically ill patients. Recoveries of the phenylcarboxylic acids from serum samples using microextraction by packed sorbent were 30-70%. The present method was linear (R2  ≥ 0.9981) over a clinically significant range of concentrations (94-2250 µg L-1/0.5-18 µM). The limits of quantification for the optimized method were 60-100 µg L-1/0.4-0.7 µM for phenylpropionic, phenyllactic, 4-hydroxybenzoic and 4-hydroxyphenylacetic acids, and 160 µg L-1/0.9-1.3 µM for benzoic, 4-hydroxyphenyllactic, homovanillic and 4-hydroxyphenylpropionic acids. The developed conditions were used to determine concentrations of the phenylcarboxylic acids in the most complicated matrix - serum samples of critically ill patients. Results were compared with liquid-liquid extraction and revealed a reduction in the time for sample preparation (45 min vs. 6 min) and serum (200 µL vs. 80 µL) volume. The combination of microextraction by packed sorbent and GC-MS methods, especially when fully automated could be a powerful tool for the clinical diagnosis of sepsis.