Is There an Adduct of Pyridine N-Oxide and Boron Trifluoride in the Gaseous State? Gas Electron Diffraction vs Mass Spectrometry.

A study of saturated vapor over the pyridine N-oxide-boron trifluoride (PyO-BF3) adduct was carried out at T = 448(5) K by a synchronous gas electron diffraction/mass spectrometry (GED/MS) experiment. Due to the absence of ions in the mass spectrum, indicating the presence of a structure with an O-B dative bond, several models of vapor composition were tested by the GED method. It was found that the dominant molecular form (up to 100%) in vapor is the PyO-BF3 adduct. Using the DFT/M06-2X/aug-cc-pVTZ method, geometric optimization of the molecular ion [PyO-BF3]+ was carried out, which showed its intrinsic instability and dissociation into a [PyO]+ cation and a BF3 molecule. This study certainly demonstrates the significant advantage of the GED method to determine the qualitative and quantitative gas-phase composition of dative-bonded adducts and other noncovalent complexes as well, whereas the interpretation of mass spectra may be ambiguous due to the possible intrinsic instability of ions containing a dative bond. The nature of the O-B bond is discussed in terms of the natural bond orbitals (NBOs) and the quantum theory of atoms in molecules (QTAIM). A comparison of structural and energetic parameters for PyO-BF3 and the previously studied BF3 adducts allows the theoretical comprehension of the nature of the O-B bond to be extended and to explain the different thermal stabilities of these compounds.

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.

Determination of Tryptophan Metabolites in Serum and Cerebrospinal Fluid Samples Using Microextraction by Packed Sorbent, Silylation and GC-MS Detection.

Indole-containing acids-tryptophan metabolites-found in serum and cerebrospinal fluid (CSF) samples of patients with diseases of the central nervous system (CNS) were determined with the use of microextraction by packed sorbent (MEPS) followed by silylation and gas chromatography-mass spectrometry (GC-MS) analysis. MEPS with the following silylation led to the reproducible formation of derivatives with an unsubstituted hydrogen ion in the indole ring, the chromatographic peaks of which are symmetric and can be used for GC-MS analysis without additional derivatization. The recoveries of analytes at the limit of quantitation (LOQ) levels were 40-80% for pooled CSF and 40-60% for serum. The limit of detection (LOD) and LOQ values were 0.2-0.4 and 0.4-0.5 µM, respectively, for both CSF and serum. The precision (the reproducibility, RSD) value of less than 20% and the accuracy (the relative error, RE) value of less than ±20% at the LOQ concentrations meet the Food and Drug Administration (FDA) recommendations. Linear correlations for all analytes were determined over a potentially clinically significant range of concentrations (0.4-10 µM for serum, R2 ≥ 0.9942, and 0.4-7 µM for CSF, R2 ≥ 0.9949). Moreover, MEPS significantly reduced the matrix effect of serum compared to liquid-liquid extraction (LLE), which was revealed in the example of reducing the amount of cholesterol and its relative compounds.

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.