Mass Spectrometric Method Research Articles

Dynamic accelerated stress test and coupled on-line analysis program to elucidate aging processes in proton exchange membrane fuel cells

The application of hydrogen proton exchange membrane fuel cells (PEMFC) in greenhouse gas emission free heavy-duty vehicles requires extremely durable PEMFC components with service lives in the range of 30,000 h. Hence suitable test and analysis methods are required that reflect realistic operation scenarios, but significantly accelerate aging. For this purpose, a dynamic accelerated stress test was developed, which is coupled with a comprehensive in-depth in-situ and ex-situ analysis program to determine the aging processes of a PEMFC membrane electrode assembly (MEA). The test comprehends dynamic cycling between low, moderate and high load, different temperature and humidity conditions as well as recovery sequences to distinguish between reversible and irreversible failure modes. All phases of the PEMFC system (i.e. solid, liquid and gaseous) are monitored on-line during aging by sophisticated electrochemical, mass spectrometric and ion chromatographic analytical methods. The structural and elemental composition of the MEA before and after the aging program (post-mortem) are investigated by X-ray fluorescence, scanning and transmission electron microscopy. This program was able to age a commercial PEMFC to end-of-life in 1000 h, while providing an accurate picture of the aging processes involved.

Spaceflight impacts xyloglucan oligosaccharide abundance in Arabidopsis thaliana root cell walls

Over the course of more than a decade, space biology investigations have consistently indicated that cell wall remodeling occurs in a variety of spaceflight-grown plants. Here, we describe a mass spectrometric method to study the fundamental composition of xyloglucan, the most abundant hemicellulose in dicot cell walls, in space-grown plants. Four representative Arabidopsis root samples, from a previously conducted spaceflight experiment - Advanced Plant EXperiment - 04 (APEX-04), were used to investigate changes in xyloglucan oligosaccharides abundances in spaceflight-grown plants compared to ground controls. In situ localized enzymatic digestions and surface sampling mass spectrometry analysis provided spatial resolution of the changes in xyloglucan oligosaccharides abundances. Overall, the results showed that oligosaccharide XXLG/XLXG and XXFG branching patterns were more abundant in the lateral roots of spaceflight-grown plants, while XXXG, XLFG, and XLFG/XLFG were more abundant in the lateral roots of ground control plants. In the primary roots, XXFG had a higher abundance in ground controls than in spaceflight plants. This methodology of analyzing the basic components of the cell wall in this paper highlights two important findings. First, that are differences in the composition of xyloglucan oligosaccharides in spaceflight root cell walls compared to ground controls and, second, most of these differences are observed in the lateral roots. Thus, the methodology described in this paper provides insights into spaceflight cell wall modifications for future investigations.

Full-length single-molecule protein fingerprinting.

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.

Identification and quantification of both isomers of hexahydrocannabinol, (9R)-hexahydrocannabinol and (9S)-hexahydrocannabinol, in three different matrices by mass spectrometry.

Hexahydrocannabinol (HHC), a compound derived from synthetic production using cannabidiol (CBD) or delta-9-tetrahydrocannabinol (Δ9 -THC), has gained recent attention due to its presence in seized materials across Europe. Sold legally in various forms, HHC poses potential health risks, particularly as a legal alternative to THC in some countries. Despite its historical description in the 1940s, limited toxicology data, pharmacological understanding, and analytical methods for HHC exist. This study proposes analytical techniques using mass spectrometry to detect, identify, and quantify (9R)-HHC and (9S)-HHC, concurrently with THC and CBD in various matrices, including oral fluid, whole blood, and seized material. Three distinct methods were employed for different matrices: GC/MS for seized material, GC/MS/MS for whole blood, and UHPLC/MS/MS for oral fluid. Methods were validated qualitatively for oral fluid with a FLOQSwab® device and quantitatively in whole blood and seized material according to Peters et al's recommendations and ICH guidelines. Validated methods were considered reliable in detecting and quantifying HHC isomers in terms of repeatability, reproducibility, and linearity with r2 systematically >0.992. These methods were applied to authentic cases, including seized materials and biological samples from traffic control (whole blood and oral fluid). In seized materials, (9R)-HHC levels ranged from 2.09% to 8.85% and (9R)-HHC/(9S)-HHC ratios varied from 1.36 to 2.68. In whole blood sample, (9R)-HHC and (9S)-HHC concentrations were, respectively, 2.38 and 1.39 ng/mL. For all analyzed samples, cannabinoids such as THC and CBD were also detected. This research contributes analytical insights into differentiating and simultaneously analyzing (9R)-HHC and (9S)-HHC, using widely applicable mass spectrometric methods. The study emphasizes the need for vigilance among toxicologists, as new semisynthetic cannabinoids continue to emerge in Europe, with potential health implications. The findings underscore the importance of reliable analytical methods for monitoring these compounds in forensic and clinical settings.

Liquid Chromatography Tandem Mass Spectrometric Method for Quantification of Margetuximab in Rat Plasma and Application to a Pharmacokinetic Study.

Margetuximab was approved for the treatment of advanced HER2+ breast cancer. A feasible analytical technique that can measure this drug was obligatory. In light of this, a novel and thoroughly validated liquid chromatographic (LC)-tandem mass spectrometric (MS/MS) approach was developed for the quantification of margetuximab in rat plasma. The liquid-liquid extraction method was used to extract the analyte from rat plasma. The analyte was separated using acetonitrile and formic acid buffer (30:70) as a mobile phase on Waters, alliance e-2695 model HPLC having Symmetry C18 column, 150 mm × 4.6 mm, 3.5-µm column. The overall runtime was 6 min at a flow rate of 1.0 ml/min. The method showed significant sensitivity and acceptable linearity over the concentration range of 6-120 ng/ml. Accuracy was within 98.51-99.92%. The intraday precision ranged between 0.41 and 8.98% CV. Also, the findings of pharmacokinetic parameters such as Cmax, tmax, AUC0-∞, AUC0-t, and half-life results of margetuximab showed that the technique was helpful for accurately measuring drug concentrations in rat plasma. The method that was developed was useful and effective for quantifying margetuximab.

LC-HRMS methods for the quantification of two artemisinin drugs and their metabolites in rat blood and plasma.

As first-line antimalarials used in the artemisinin combination therapy, artemisinin drugs exert their action inside red blood cells. However, the blood pharmacokinetic characteristics of artemisinin drugs have not been fully revealed owing to their built-in chemical instability initiated by Fe2+ released from hemoglobin, with limited information on their metabolites. In this study, liquid chromatography tandem high-resolution mass spectrometric (LC-HRMS) methods were developed for the quantification of two representative artemisinin drugs (artemisinin, ART; dihydroartemisinin, DHA) and their respective metabolite (deoxyartemisinin, D-ART; dihydroartemisinin glucuronide, DHA-Glu) in rat blood/plasma. The blood samples were pretreated with the stabilizer (0.4m potassium dichromate and 3% EDTA-2Na). The methods displayed excellent specificity, linearity, accuracy and precision for ART (17.7-709.2nm) and its metabolite D-ART (18.8-751.9nm), and the linear range was 40.0-4,000.0nm for both DHA and DHA-Glu. The methods were successfully applied to the pharmacokinetic studies of ART and DHA in rats. The blood-to-plasma ratio was 0.8-1.5 for ART, 1.0-1.5 for D-ART, 1.2-2.2 for DHA and 0.9-1.3 for DHA-Glu, which was time dependent. The results indicated that artemisinin drugs and their metabolites showed a high but different blood-to-plasma ratio, which should be considered when optimizating their dosing regimens or evaluating their clinical outcomes.

Quantitative mass spectrometry with ¹⁸O labelling as an alternative approach for determining protease activity: an example of trypsin

SCIENTIFIC RELEVANCE. In the quality control of proteolytic enzyme components of medicinal products, the activity of proteases is determined by spectrophotometry, which involves mea­suring the amidase or esterase activity using a synthetic substrate and the proteolytic activity using the Anson method. These methods require special substrates and have low sensitivity; their specificity may be insufficient, which may lead to serious errors. Quantitative mass spectrometry is an alternative approach to protease activity assays, which involves adding an isotope-labelled peptide to hydrolysates of the test enzyme. This approach allows determining the activity of proteases, notably, by the hydrolysis of specific peptide bonds, while simulta­neously confirming the identity and specificity of the test sample. Quantitative mass spectrometry has high sensitivity and does not require special substrates.AIM. This study aimed to investigate the possibility of enzymatic activity assay and enzyme identification by quantitative mass spectrometry with ¹⁸O labelling through an example of trypsin with casein.MATERIALS AND METHODS. The study used trypsin, casein, and H₂¹⁸O (Izotop, Russia). Peptide separation was performed using an Agilent 1100 HPLC system; mass spectra were obtained using a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer. Quantitative mass spectrometry was performed using a standard peptide, which was obtained from casein by tryptic digestion and HPLC purification. For ¹⁸O labelling, the authors dried the peptide and incubated it in H₂¹⁸О water. The quantitative analysis of the product was carried out using MALDI-TOF mass spectrometry. The authors used quantitative mass spectrometry with ¹⁸O labelling to determine enzymatic activity and calculate the Michaelis constant (KM).RESULTS. Following the tryptic digestion of casein, the authors identified the fragments corre­sponding to casein chains. The authors produced the isotope-labelled standard peptide and calculated its concentration using mass spectrometry. The authors determined the rate of casein digestion by trypsin and calculated the KM for trypsin, which was 13.65±0.60 μM. The standard deviation for repeated measurements showed that the mass-spectrometric method had a lower error of measurement than the spectrophotometric method. The sensitivity threshold for the mass-spectrometric method was 0.50±0.08 μM.CONCLUSIONS. The results obtained with trypsin confirm the possibility of enzymatic activity determination by the proposed method of quantitative mass spectrometry with ¹⁸O labelling. According to the sensitivity evaluation results, this method can be used for the simultaneous determination of enzyme activity, identity, and specificity. The proposed mass spectrometry approach is universal, it does not require expensive materials and reagents, and it can be easily adapted to determine the activity of virtually any protease.

Contamination of Kazakhstan cheeses originating from Escherichia coli and its resistance to antimicrobial drugs

Background and Aim: Escherichia coli, a commensal intestine bacterium of vertebrates, is widely distributed in the environment and indicates the microbiological quality of food products in relation to coliforms. In addition, virulent strains, particularly E. coli O157:H7, cause outbreaks of toxic infections caused by consuming dairy products. Because food safety studies regarding E. coli have not been conducted in Central Asia, this research aimed to study the characteristics of contamination, microbiological and genotypic properties, and resistance to antimicrobial agents of E. coli strains that contaminate various types of commercialized cheeses originating from Kazakhstan. Materials and Methods: In retail outlets, 207 samples of three types of cheese produced by 22 industrial and eight small enterprises in the central, eastern, southern, and northern regions of Kazakhstan were selected in 2020–2023. E. coli contamination was examined using standard microbiological, mass spectrometric, and molecular genetic methods. The discodiffuse European Committee on Antimicrobial Susceptibility Testing method was used to test the resistance of the identified E. coli isolates (65/207; 31.4%) to 20 antibacterial drugs. The Shiga toxin-producing E. coli (VT1 and VT2) and E. coli O157:H7 (eae) genes were investigated in all E. coli isolates using multiplex polymerase chain reaction. Results: An average of 31.4% samples of commercial Kazakhstani cheeses of various types were found to be contaminated with E. coli in almost all geographical regions of Kazakhstan, regardless of the productivity of the dairy enterprises. Soft cheeses produced by small farms (80% of samples) packaged at the retail site (100%) were the most contaminated with E. coli. The microbiological index (colony-forming unit/g) was unsatisfactory and unsuitable in 6.2% of such cheese samples. For the first time in Central Asia, the enteropathogenic strain E. coli O157:H7 was detected in 0.5% of cheese samples. E. coli isolates from cheese samples were resistant to 65% of antibacterial drugs and contained resistance genes to β-lactams, sulfonamides, and quinolones groups. At the same time, 25% of the E. coli isolates were multi-resistant to three or more antimicrobial agents. Conclusion: The high level of contamination caused by multi-antibiotic resistant E. coli strains, including pathogenic pathogens, poses a risk to public health and highlights the need for further research on the monitoring and control of coliform enteropathogens in food products. Keywords: antibiotic resistance, Escherichia coli O157:H7, Kazakhstan cheese, microbial contamination.

Reliable biological indicator identification and evaluation of tobacco-derived nicotine using an ultra-sensitive gas chromatography-tandem mass spectrometric method.

Several countries have exempted synthetic nicotine from existing regulatory frameworks, resulting in the widespread substitution of synthetic nicotine (SN) in almost all e-cigarette products available. However, it remains uncertain whether the purported synthetic nicotine is indeed genuine SN. There is a need to develop biological indicators and an analytical method that more clearly distinguishes between the two sources. Impurities in neat tobacco-derived nicotine (TDN) were characterized and identified through non-targeted and targeted analysis. Gas chromatography-tandem mass spectrometry (GC-MS/MS) conditions were optimized for detecting biological indicators in e-cigarette products. Nine tobacco-related alkaloids were identified and selected as biological indicators for TDN. A liquid-liquid extraction and GC-MS/MS quantitative method were developed to detect nine biological indicators in e-cigarette products with the limit of quantification ranging from 0.2 to 4.2µg L-1 using 0.5mL of e-liquid. This method was applied to 50 e-cigarette brands purchased in the Korean market. The developed method was able to easily and accurately identify the origin of nicotine even using a small amount of e-liquid sample. It is expected that effective e-cigarette regulation will be possible if the nicotine biological indicator and high-sensitivity analysis method developed in this study are used.

Multiscale Investigation of Sodium‐Ion Battery Anodes: Analytical Techniques and Applications

AbstractThe anode/electrolyte interface behavior, and by extension, the overall cell performance of sodium‐ion batteries is determined by a complex interaction of processes that occur at all components of the electrochemical cell across a wide range of size‐ and timescales. Single‐scale studies may provide incomplete insights, as they cannot capture the full picture of this complex and intertwined behavior. Broad, multiscale studies are essential to elucidate these processes. Within this perspectives article, several analytical and theoretical techniques are introduced, and described how they can be combined to provide a more complete and comprehensive understanding of sodium‐ion battery (SIB) performance throughout its lifetime, with a special focus on the interfaces of hard carbon anodes. These methods target various length‐ and time scales, ranging from micro to nano, from cell level to atomistic structures, and account for a broad spectrum of physical and (electro)chemical characteristics. Specifically, how mass spectrometric, microscopic, spectroscopic, electrochemical, thermodynamic, and physical methods can be employed to obtain the various types of information required to understand battery behavior will be explored. Ways are then discussed how these methods can be coupled together in order to elucidate the multiscale phenomena at the anode interface and develop a holistic understanding of their relationship to overall sodium‐ion battery function.

High‐temperature Mass Spectrometric Study and Modeling of the Thermodynamic Properties of the TiO2‐SiO2‐ZrO2 System

AbstractDevelopment of the advanced materials based on the TiO2‐SiO2‐ZrO2 system is hindered by lack of the information on its thermodynamic properties and phase equilibria. In the present study, the samples in the TiO2‐SiO2‐ZrO2 system were prepared by solid‐state synthesis and analyzed by X‐ray microanalysis, fluorescence, and diffraction techniques. The thermodynamic properties in this system were evaluated by Knudsen effusion mass spectrometric (KEMS) method and optimized within the generalized lattice theory of associated solutions (GLTAS). The main vapor species over the silica‐containing samples vaporized from the tungsten cell at the temperature 1973 K were SiO, O, WO2, and WO3. The vapor over the samples in the TiO2‐ZrO2 system at 2356 K was composed mainly of TiO, TiO2, O, WO2, and WO3. The activity of components in the samples and the partial pressures of the identified vapor species were determined. At 1973 K, the TiO2‐SiO2‐ZrO2 system is characterized by positive deviations from the ideality. The observed weak dependence of the SiO2 activity on the SiO2 content suggests the presence of a broad field of immiscibility on the phase diagram in the concentration range 40–100 mol . % SiO2. The thermodynamic data on the TiO2‐SiO2‐ZrO2 system obtained by KEMS can be of great value for calculation of the phase equilibria.

Trace detection of perfluorooctanoic acid and perfluorooctane sulfonate in surface sediments using a liquid chromatograph coupled to an electrospray ionization single quadrupole mass spectrometer (LC-ESI–Q-MS)

Poly- and perfluoroalkyl substances (PFAS) are among the pollutants of environmental concern besides pharmaceuticals and personal care products, microplastics, and other persistent organic pollutants. Of all the different PFASs reportedly present in the environment, the most researched are perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS). In this study, these pollutants were detected using a precise, selective, and sensitive liquid chromatographic-electrospray ionization single quadrupole mass spectrometric (LC-ESI–Q-MS) method within a linear range of 0.002–0.02 μg/mL. The method developed was thereafter validated according to the ICH validation parameters. This method was used to monitor PFOA and PFOS in selected surface sediments collected within the environs of two prominent rivers in the KwaZulu-Natal province of South Africa: Umgeni and Palmiet Rivers. While the PFOA levels varied from not detected to 1.490 ng/g dw, and PFOS levels ranged from 0.206 ng/g dw to 0.426 ng/g dw, the total PFOA and PFOS concentration (∑ PFOA and ∑ PFOS) in the surface sediment samples collected were 7.50 ng/g dw and 2.79 ng/g dw, respectively. The percent positive occurrence of PFOA and PFOS in all the tested sediment samples was 81.8 % and 100 %, respectively. This study becomes an eye opener to the commencement of research works with respect to developing remediation strategies for the removal of PFAS in the environment, especially the African environment.

Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches.

Traditional studies of cellular metabolism have relied on the use of radioisotopes. These have clear disadvantages associated with safety and waste generation. Furthermore, detection of the labeled species by scintillation counting provides only a quantification of its presence or absence. The use of stable isotopes, by contrast, allows the application of powerful, orthogonal spectroscopic approaches such as nuclear magnetic resonance spectroscopy (NMR) and various mass spectrometric methods. Using stable isotope labeling to study heme metabolism requires integrating methods for (a) generating the heme in labeled forms, (b) cultivating and quantifying the organism of choice in chemically defined media, to which labeled compounds can be added, (c) recovering cellular components and/or spent growth media, and (d) analyzing these materials for the labeled species using spectroscopic and mass spectrometric methods. These methods are summarized here in the context of Bacteroides thetaiotaomicron, a generallynonpathogenic anaerobe and heme auxotroph.

ХАРАКТЕРИСТИКА И ВЛИЯНИЕ МИКРОКАПСУЛЯЦИИ НА СТАБИЛЬНОСТЬ БИОПЕПТИДА В МОДЕЛИ IN VITRO

One of the ways to prevent the development of oxidative stress is the oral use of bioactive peptides. To ensure the targeted release of biologically active substances in the body, they are microcapsulated. The aim of the research is to characterize, predict, evaluate the antioxidant properties of a biopeptide and study the effect of microcapsulation on its stability in a model of the gastrointestinal tract. The molecular mass distribution of the peptide was evaluated by mass spectrometric method. Identification and characterization of the peptide was carried out using Protein NCBI and ARD databases. The antioxidant activity of the peptide was determined by DPPH, ABTS and FRAP methods. The microencapsulation of the peptide was carried out in a pseudo-boiling layer of 10 % maltodextrin solution. As a result of research, a peptide was isolated from trypsin hydrolysate of cow colostrum, its molecular weight, amino acid sequence, charge, hydrophobicity, isoelectric point, two- and three-dimensional structure were determined. Based on the obtained physicochemical characteristics of the peptide, a hypothesis about its antioxidant properties has been proposed, which has been confirmed in in vitro studies. It is proved that the microcapsulation of the peptide into a protective layer of maltodextrin ensures its stability in the model of the oral cavity and stomach and targeted release in the intestine in vitro.

On the extension of the use of a standard operating procedure for nicotine, glycerol and propylene glycol analysis in e-liquids using mass spectrometry.

Standard operating procedures (SOP), accessible to several laboratories, are essential for product verification. EU-JATC (European-Joint Action on Tobacco Control) SOP and the WHO TobLabNet (World Health Organization Tobacco Laboratories Network) SOP (SOP11) are available standard methodologies to measure nicotine, glycerol, and propylene glycol, and propose mass spectrometer (MS) as an alternative method to flame ionization detector (FID). This study conducted a comparison between FID and MS concentration results, following the MS method described in SOP11. In May 2020, five test e-liquids in replicates (A-E) were prepared at the Istituto di Ricerche Farmacologiche Mario Negri and sent, with SOP 11, validation document and results datasheet to 32 different laboratories all over the world from WHO TobLabNet and EU-JATC (18 from JATC, ten from WHO TobLabNet and four academic laboratories). Among thirty-two independent laboratories that participated in the study, results were received from 30 laboratories. The e-liquids analyses, using the two approaches, were compared. Of the 30 laboratories surveyed, 21 utilized the FID approach exclusively, 7 opted for MS detection, and 2 employed both methods. The findings demonstrated that the gas chromatography-mass spectrometry (GC-MS) method offers comparable analytical capabilities regarding accuracy and precision for nicotine, glycerol, and propylene glycol to the FID approach. Through Pearson's correlation test with r≃1 showing a positive correlation between GC-FID and GC-MS data, and the Student's t-test, no significant differences between the two approaches were revealed, showing p>0.005 for almost all three analytes in all samples. This study indicates that it is possible to apply the available EU-JATC SOP and the WHO TobLabNet SOP11 even in laboratories that do not have access to an FID, for example, to analyze flavors, trace compounds or carcinogenic, mutagenic, or toxic for reproduction (CMR) in electronic cigarette liquids.

Quantification of asciminib by LC-MS/MS method in human plasma: Validation and stability studies

An accurate, sensitive, and linear liquid chromatographic tandem mass spectrometric method was developed for the quantification of asciminib in human plasma. Voriconazole, internal standard (IS), and asciminib were isolated from the plasma samples by C18-cartridges. Chromatographic isolation was processed with Atlantis/dC18 (2.1 × 100 mm, 3 μm) stationary phase and a mobile solvent system of formic acid (0.1%) and methyl alcohol (20:80, v:v), conveyed at a 0.7 ml/minute flowing rate. Analytes were quantified by ionization in a positive approach with electrospray ionization at the mass transitions of (m/z): asciminib, 450.11/239.09, and voriconazole (IS), 350.3/281.1. No interference by constituents of plasma blank or other components was detected. The association between asciminib concentration levels and their respective peak response fractions to voriconazole was rectilinear between 39.0 and 1,586.0 ng/ml. Intraday and interday precisions were ≤4.79% for asciminib. The interday bias was between −4.28% and 5.76%, and the intraday bias was −3.75% and 4.53%. The mean measured extraction recovery of asciminib was 99.06%. The recovery of IS was 98.34%. Asciminib was subjected to long-term, bench-top, freeze-thaw, short-term stability, dry extract, auto-sampling, and stock solution stability at Low quality control and High quality control levels, and it was stable at all these conditions. The established technique can be utilized for the regular quantification of asciminib in plasma samples in industries, forensic laboratories, and clinical research organizations.

A sensitive liquid chromatography tandem mass spectrometric method development and validation for ribociclib and its formulation

This study implies to development of a sensitive liquid chromatography tandem mass spectrometry method. Further using this method ribociclib can be estimated in the drug formulation. Ribociclib was recently approved by the United States Food and Drug Administration in the year 2017 for metastatic and advanced breast cancer, which is used for resistant patients to endocrine therapy and it targets cyclin-dependent kinase 4 and 6 inhibitors. The stationary phase was Phenomenex C18 (50 × 4.6 mm, 3 microns), with a mobile phase combination of acetonitrile and 10 mM ammonium acetate (90:10 v/v ratio) with a flow of 0.7 ml/minutes. Ribociclib was detected using liquid chromatography-tandem mass spectrometry in positive transition at m/z 435.0. The linearity ranges between 5 and 100 ng/ml. The Ribociclib correlation coefficient (R2) was linear, with an R2 value of 0.9991. The drug had a retention time (RT) of 1.022 minutes, and a total run time of 3 minutes. According to the literature survey, there is a limited isocratic method for the selected drug hence, we developed an isocratic which is a quick, accurate, and efficient method for determining ribociclib and its formulation.

Tannic acids and proanthocyanidins in tea inhibit SARS-CoV-2 variants infection.

The COVID-19 pandemic has caused hundreds million cases and millions death as well as continues to infect human life in the world since late of 2019. The breakthrough infection caused from mutation of SARS-CoV-2 is rising even the vaccinated population has been increasing. Currently, the severe threat posed by SARS-CoV-2 has been alleviated worldwide, and the situation has transitioned to coexisting with the virus. The dietary food with antiviral activities may improve to prevent virus infection for living with COVID-19 pandemic. Teas containing enriched phenolic ingredients such as tannins have been reported to be antitumor agents as well as be good inhibitors for coronavirus. This study developed a highly sensitive and selective ultra-high performance liquid chromatography-high resolution mass spectrometric method for quantification of tannic acids, a hydrolysable tannin, and proanthocyanidins, a condense tannin, in teas with different levels of fermentation. The in vitro pseudoviral particles (Vpp) infection assay was used to evaluate the inhibition activities of various teas. The results of current research demonstrate that the tannins in teas are effective inhibitors against infection of SARS-CoV-2 and its variants.

Liquid Chromatography with Tandem Mass Spectrometric Method Development and Validation Study for the Estimation of N-Nitrosodimethylamine in Ranitidine Tablets

Liquid Chromatography with Tandem Mass Spectrometric Method Development and Validation Study for the Estimation of N-Nitrosodimethylamine in Ranitidine Tablets

Development of a rapid mass spectrometric method for the analysis of ten-eleven translocation enzymes.

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.