Liquid Chromatography-mass Spectrometry Method Research Articles

Metformin drugs under simulated gastric conditions can generate high nitrite-dependent levels of N-nitrosodimethylamine

N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), group 2A carcinogens, were detected in finished drug products, including metformin, ranitidine, sartans and other drugs which caused multiple recalls in the USA and Europe. Important studies also reported the formation of NDMA when ranitidine and nitrite were added to simulated gastric fluid. Our objective was to screen finished drug products from Europe and USA for nitrosamine impurities and investigate the formation of NDMA in metformin finished drug products when added to simulated gastric fluid. One dosage unit of 30 different commercially available drugs, including metformin, sartans, and ranitidine were tested for NDMA, NDEA, and dimethylformamide (DMF) impurities, using a liquid chromatography-mass spectrometry (LC–MS) method. Then, 6 metformin finished drug products were tested in stomach conditions for 2 h at 37 °C in a 100 mL solution with a pH of 2.5 and different nitrite concentrations (40, 10, 1, 0.1 mM) and tested for NDMA, and DMF using LC–MS. We measured NDMA, NDEA, and DMF in 30 finished drug products. NDMA and DMF were quantified for metformin drug products in simulated gastric fluid with different nitrite concentrations. None of the 30 drugs showed concerning levels of NDMA, NDEA, or DMF when tested as single tablets. However, when metformin tablets are added to simulated gastric fluid solutions with high nitrite concentrations (40 mM and 10 mM), NDMA can reach amounts of thousands of nanograms per tablet. At the closest concentration to physiologic conditions we used, 1 mM, NDMA is still present in the hundreds of nanograms in some metformin products. In this in vitro study, nitrite concentration had a very important effect on NDMA quantification in metformin tablets added to simulated gastric fluid. 1 mM nitrite caused an increase above the acceptable daily intake set by the U.S. Food and Drug Administration (FDA) for some of the metformin drugs. 10 mM, 40 mM nitrite solutions generated NDMA amounts exceeding by more than a hundred times the acceptable daily intake set by the FDA of 96 nanograms. These findings suggest that metformin can react with nitrite in gastric-like conditions and generate NDMA. Thus, patients taking metformin could be exposed to NDMA when high nitrite levels are present in their stomach, and we recommend including a statement within the Patient Package Inserts/Instructions for use.

Identification of brain-enriched proteins in CSF as biomarkers of relapsing remitting multiple sclerosis

BackgroundMultiple sclerosis (MS) is a clinically and biologically heterogenous disease with currently unpredictable progression and relapse. After the development and success of neurofilament as a cerebrospinal fluid (CSF) biomarker, there is reinvigorated interest in identifying other markers of or contributors to disease. The objective of this study is to probe the predictive potential of a panel of brain-enriched proteins on MS disease progression and subtype.MethodsThis study includes 40 individuals with MS and 14 headache controls. The MS cohort consists of 20 relapsing remitting (RR) and 20 primary progressive (PP) patients. The CSF of all individuals was analyzed for 63 brain enriched proteins using a method of liquid-chromatography tandem mass spectrometry. Wilcoxon rank sum test, Kruskal-Wallis one-way ANOVA, logistic regression, and Pearson correlation were used to refine the list of candidates by comparing relative protein concentrations as well as relation to known imaging and molecular biomarkers.ResultsWe report 30 proteins with some relevance to disease, clinical subtype, or severity. Strikingly, we observed widespread protein depletion in the disease CSF as compared to control. We identified numerous markers of relapsing disease, including KLK6 (kallikrein 6, OR = 0.367, p < 0.05), which may be driven by active disease as defined by MRI enhancing lesions. Other oligodendrocyte-enriched proteins also appeared at reduced levels in relapsing disease, namely CNDP1 (carnosine dipeptidase 1), LINGO1 (leucine rich repeat and Immunoglobin-like domain-containing protein 1), MAG (myelin associated glycoprotein), and MOG (myelin oligodendrocyte glycoprotein). Finally, we identified three proteins—CNDP1, APLP1 (amyloid beta precursor like protein 1), and OLFM1 (olfactomedin 1)—that were statistically different in relapsing vs. progressive disease raising the potential for use as an early biomarker to discriminate clinical subtype.ConclusionsWe illustrate the utility of targeted mass spectrometry in generating potential targets for future biomarker studies and highlight reductions in brain-enriched proteins as markers of the relapsing remitting disease stage.

A ng/L Level LC-MS Method Using Membrane SPE as Sampling Technology: Determination of Nine Halobenzoquinones in Potable Water.

A promising method was established for the determination of nine halobenzoquinones (HBQs) in potable water by membrane solid-phase extraction (MSPE) pretreatment and the liquid chromatography-mass spectrometry (LC-MS) method. A 500 mL water sample was taken for enrichment by the SDB-RPS membrane, which was previously activated by methanol and ultrapure water. The sample was eluted with methanol and re-dissolved with the initial mobile phase after nitrogen blowing. Then, it was detected in negative ion mode using the working curve, and HBQs were quantified by the external standard method. The linearity was satisfactory in the concentration range of 4-1000 ng/L, with correlation coefficients of 0.9963~0.9994. The recoveries were 73.5~126.6% at three spiked levels, with relative standard deviations (RSDs) of 6.8~15.5%. The limits of detection (LOD, S/N = 3) values were 0.1~0.7 ng/L. The results demonstrate that the MSPE-LC-MS method is reliable, rapid, and sensitive for the simultaneous analysis of nine HBPs in potable water.

Revealing the effects and mechanism of wine processing on Corni Fructus using chemical characterization integrated with multi-dimensional analyses

Corni fructus (CF) is always subjected to wine processing before prescription in clinic, for an enhancing effect of nourishing liver and kidney. While, the underlying mechanism for this processing on CF remains obscure. In this study, a sensitive ultra-high-performance liquid chromatography mass spectrometry (UPLC-MS/MS) method combined multi-dimensional analyses was established to monitor chemical characterizations of raw and wine-processed CF (WCF) and hence reveal the effects and underlying mechanism of wine processing on CF. As indicated, a total of 216 compounds were tentatively identified, including 98 structurally complex and variable home/hetero-polymers, that were composed of iridoid glucosides, gallic acids, caffeic acid and/or 5-HMF. Interestingly, 53 of these compounds probably characterized potential novel, including 35 iridoid glucosides or their dimers, 9 iridoid glucoside-gallic acid dimers, 7 gallic acids derivatives and 2 gallic acid-caffeic acid dimers, which provides ideas for natural product researchers. Meanwhile, the multi-dimensional analyses including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and linear regression analysis were used to explore the differences between CF and WCF. The results showed that 23 compounds as chemical markers greatly contributing to the distinction were screened out, and 3 of which (7α/β-O-ethyl-morroniside, gallic acid and 5-HMF) in WCF indicated an increasing trend in intensities in relative to those in CF. Additionally, linear regression analysis showed that in WCF 53 compounds exhibited an increasing in intensities, while 132 ones did a decreasing trend, compared with those in CF. As our investigation demonstrated, acetal reaction of morroniside, ester hydrolysis in different organic acid derivatives as well as glycoside bond cleavage during wine processing probably resulted in the distinctions. The findings of this study provide a further understanding of the effect and mechanism of wine processing on CF.

Implementation of a LC-MS based multi-attribute method (MAM) and intact multi-attribute method (iMAM) workflow for the characterisation of a GLP-Fc fusion protein

Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography−mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level.

A new approach towards highly sensitive detection of endogenous N-acetylaspartic acid, N-acetylglutamic acid, and N-acetylaspartylglutamic acid in brain tissues based on strong anion exchange monolith microextraction coupled with UHPLC-MS/MS.

Anovel in-tube solid-phase microextraction coupled withan ultra-high performance liquid chromatography-mass spectrometry method has been established for simultaneous quantification of three crucial brain biomarkers N-acetylaspartic acid (NAA), N-acetylglutamic acid (NAG), and N-acetylaspartylglutamic acid (NAAG). A polymer monolith with quaternary ammonium as the functional group was designed and exhibited efficient enrichment of target analytes through strong anion exchange interaction. Under the optimized conditions, the proposed method displayed wide linear ranges (0.1-80 nM for NAA and NAG, 0.2-160 nM for NAAG) with good precision (RSDs were lower than 15%) and low limits of detection (0.019-0.052 nM), which is by far the most sensitive approach for NAA, NAG, and NAAG determination. Furthermore, this approach has been applied to measure the target analytes in mouse brain samples, and endogenous NAA, NAG, and NAAG were successfully detected and quantified from only around 5mg of cerebral cortex, cerebellum, and hippocampus. Compared with existing methods, the newly developed method in the current study provides highest sensitivity and lowest sample consumption for NAA, NAG, and NAAG measurements, which would potentially be utilized in determining and tracking these meaningful brain biomarkers in diseases or treatment processes, benefiting the investigations of pathophysiology and treatment of brain disorders.

Liquid Chromatography-Mass Spectrometry Analytical Methods for the Quantitation of p-Cresol Sulfate and Indoxyl Sulfate in Human Matrices: Biological Applications and Diagnostic Potentials.

Indoxyl sulfate (IxS) and p-cresyl sulfate (pCS) are toxic uremic compounds with documented pathological outcomes. This review critically and comprehensively analyzes the available liquid chromatography-mass spectrometry methods quantifying IxS and pCS in human matrices and the biological applications of these validated assays. Embase, Medline, PubMed, Scopus, and Web of Science were searched until December 2023 to identify assays with complete analytical and validation data (N = 23). Subsequently, citation analysis with PubMed and Scopus was utilized to identify the biological applications for these assays (N = 45). The extraction methods, mobile phase compositions, chromatography, and ionization methods were evaluated with respect to overall assay performance (e.g., sensitivity, separation, interference). Most of the assays focused on human serum/plasma, utilizing acetonitrile or methanol (with ammonium acetate/formate or formic/acetic acid), liquid-liquid extraction, reverse phase (e.g., C18) chromatography, and gradient elution for analyte separation. Mass spectrometry conditions were also consistent in the identified papers, with negative electrospray ionization, select multiple reaction monitoring transitions and deuterated internal standards being the most common approaches. The validated biological applications indicated IxS and/or pCS were correlated with renal disease progression and cardiovascular outcomes, with limited data on central nervous system disorders. Methods for reducing IxS and/or pCS concentrations were also identified (e.g., drugs, natural products, diet, dialysis, transplantation) where inconsistent findings have been reported. The clinical monitoring of IxS and pCS is gaining significant interest, and this review will serve as a useful compendium for scientists and clinicians.

Formation of Cysteine Adducts with Chlorogenic Acid in Coffee Beans.

The post-harvest processing of coffee beans leads to a wide range of reactions involving proteins. The formation of crosslinks between proteins and phenolic compounds present in high concentrations of coffee beans represents one of the most challenging and still not fully characterized reactions. The aim of this work was to assess the presence of products from such reactions in coffee samples, focusing on the adducts between cysteine and chlorogenic acids (CQAs). For this purpose, 19 green and 15 roasted coffee samples of the Coffea arabica, Coffea canephora, and Coffea liberica varieties were selected for this study and basically characterized. Then, targeted liquid chromatography mass spectrometry (LC-MS/MS) methods were developed to assess the formation of adducts between CQA and cysteine, glutathione, and N-acetylcysteine as the amino acid and peptide models, and quantified such adducts in coffee samples. The results of the characterization showed a heterogeneous distribution of the protein content (8.7-14.6%), caffeine (0.57-2.62 g/100 g), and antioxidant capacity (2-4.5 g ascorbic acid/100 g) in Arabica, Canephora, and Liberica samples. Glutamic acid, arginine, and proline were found to be the major amino acids, while 5-CQA (38-76%), 3-CQA (4-13%), and 4-CQA (4-13%) were the most abundant CQA derivatives of all coffee varieties. The model experiments for adduct formation demonstrated that cysteine binds to CQA via thiol groups and 5-CQA initially isomerizes to 3- and 4-CQA, depending on the conditions, allowing cysteine to bind to two different sites on 3-, 4- or 5-CQA molecules, thus, forming six different Cys-CQA adducts with m/z 476. The reaction was more favored at pH 9, and the adducts proved to be stable up to 90 °C for 10 min and up to 28 days at room temperature. The relative quantification of adducts showed peak area values ranging from 1100 to 3000 in green coffee bean samples, while no adducts were detected in roasted coffee beans. Overall, this work was the first attempt to demonstrate the presence of Cys-CQA adducts in coffee beans and paves the way for further investigations of such adduct formation at the protein level.

Simultaneous quantification of five antiretrovirals in human tissues using ultra-high performance liquid chromatography-tandem mass spectrometry methods for therapeutic drug monitoring at the sites of action

Although antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection to suppress virus in the blood, HIV persists in tissues. HIV persistence in the tissues is due to numerous factors, and one of those factors are antiretroviral (ARV) concentrations. ARV concentrations in tissues must be adequate to suppress HIV at the sites of action. While therapeutic drug monitoring in the plasma is well-known, drug monitoring in the tissues provides local assessments of adequate ARV exposure to prevent localized HIV resistance formation. Towards these efforts, we validated an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method in human tissues (cervical, rectal, and vaginal tissues) for the simultaneous quantification of five ARVs: bictegravir, cabotegravir, dolutegravir, doravirine, and raltegravir. For this assay, protein precipitation with acetonitrile with stable, isotopically-labeled internal standards followed by supernatant pre-concentration was performed. Analyte separation was accomplished using a multistep UPLC gradient mixture of 0.1 % formic acid in water (A) and acetonitrile (B) with a Waters Cortecs T3 (2.1x100 mm) column. The assay was extensively validated as per the United States Food and Drug Administration Bioanalytical Method Validation Guidance over a clinically observed range (0.05–50 ng/mL) with superb linearity (R2 > 0.99 across all ARVs). The assay run time was 8.5 min. This analytical method achieves appropriate performance of trueness (85.5–107.4 %), repeatability, and precision (CV < 15 %). Our method will be employed for the therapeutic monitoring of guideline-recommended ARVs in human tissues for monitoring therapeutic efficacy in HIV treatment and prevention research efforts.

Alkylresorcinol detection and identification in archaeological pottery using ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry.

Alkylresorcinols (AR) are cereal-specific biomarkers and have recently been found in archaeological pots. However, their low concentrations and high susceptibility to degradation make them difficult to detect using conventional gas chromatography mass spectrometry (GC/MS). Here we describe the development of a more sensitive liquid chromatography mass spectrometry (LC/MS) method to detect these compounds. A method based on the use of ultra-high-performance liquid chromatography (UHPLC) coupled to an Orbitrap mass analyser was established and validated for the detection of low-concentration ARs in pottery. During the preliminary experiments, UHPLC-Q/Orbitrap MS (ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry) was demonstrated to be more sensitive, and a wide range of AR homologues in cereal extracts were detected, unlike UHPLC-QTOFMS (ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry) and GC/MS. The developed method was utilised to profile AR homologue distribution in modern cereal samples and reanalyse AR-containing pots from the archaeological site of Must Farm. A highly sensitive LC/MS method with a limit of detection (LOD) of 0.02 μg/g and a limit of quantification (LOQ) of 0.06 μg/g was used to profile ARs in five modern cereal grains. The obtained LOD is 250 times lower than that obtained using the conventional GC/MS approach. AR 21:0 was the most abundant homologue in all four Triticum spp.-einkorn, emmer, Khorasan wheat and common wheat. Meanwhile, AR 25:0 was the predominant homologue in barley, potentially enabling differentiation between wheat and barley. The developed LC/MS-based method was successfully used to analyse ARs extracted from Must Farm potsherds and identified the cereal species most likely processed in the pots-emmer wheat. The described method offers an alternative and more sensitive approach for detecting and identifying ARs in ancient pottery. It has been successfully utilised to detect AR homologues in archaeological samples and discriminate which cereal species-wheat and barley-were processed in the pots.

Potential Role of Lauric Acid in Milk Fat Synthesis in Chinese Holstein Cows Based on Integrated Analysis of Ruminal Microbiome and Metabolome.

The composition and metabolic profile of the ruminal microbiome have an impact on milk composition. To unravel the ruminal microbiome and metabolome affecting milk fat synthesis in dairy cows, 16S rRNA and internal transcribed spacer (ITS) gene sequencing, as well as ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) methods were used to investigate the significant differences in ruminal bacterial and fungal communities as well as metabolome among Chinese Holstein cows with contrasting milk fat contents under the same diet (H-MF 5.82 ± 0.41% vs. L-MF 3.60 ± 0.12%). Another objective was to culture bovine mammary epithelial cells (BMECs) to assess the effect of metabolites on lipid metabolism. Results showed that the acetate-to-propionate ratio and xylanase activity in ruminal fluid were both higher in H-MF. Microbiome sequencing identified 10 types of bacteria and four types of fungi differently abundant at the genus level. Metabolomics analysis indicated 11 different ruminal metabolites between the two groups, the majority of which were lipids and organic acids. Among these, lauric acid (LA) was enriched in fatty acid biosynthesis with its concentration in milk fat of H-MF cows being greater (217 vs. 156 mg per 100 g milk), thus, it was selected for an in vitro study with BMECs. Exogenous LA led to a marked increase in intracellular triglyceride (TG) content and lipid droplet formation, and it upregulated the mRNA abundance of fatty acid uptake and activation (CD36 and ACSL1), TG synthesis (DGAT1, DGAT2 and GPAM), and transcriptional regulation (SREBP1) genes. Taken together, the greater relative abundance of xylan-fermenting bacteria and fungi, and lower abundance of bacteria suppressing short-chain fatty acid-producing bacteria or participating in fatty acid hydrogenation altered lipids and organic acids in the rumen of dairy cows. In BMECs, LA altered the expression of genes involved in lipid metabolism in mammary cells, ultimately promoting milk fat synthesis. Thus, it appears that this fatty acid plays a key role in milk fat synthesis.

Comprehensive Glycosylation Characterization of Recombinant Human Erythropoietin by Electron-Activated Dissociation Mass Spectrometry.

Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy, which has three N-glycosylation sites and one O-glycosylation site. It contains a variety of different glycosylation modifications, such as sialyation, O-acetylation on sialic acids, etc., which causes a big challenge for the glycosylation analysis of rhEPO. In this study, a liquid chromatography-mass spectrometry (LC-MS) method combined with electron-activated dissociation (EAD) technology was used in qualitative and quantitative characterization of rhEPO N-glycosylation and O-glycosylation in just one injection. The usage of EAD not only generated abundant MS/MS fragment ions of glycopeptides and improved the MS/MS sequence coverage but also preserved the glycan structures in the MS/MS fragment ions and the integrity of the glycosidic bond between the glycans and peptides. Three N-glycosylation sites (N24, N38, and N83) and one O-glycosylation site (S126) of rhEPO samples were successfully identified. Among them, the glycosylation ratios of N24, N38, and N83 sites were 82.7%, 100%, and 100% respectively, and 15, 10, and 12 different N-glycans could be identified at the glycopeptide level. The total average number of sialic acids, N-hydroxyacetylneuraminoic acid, and O-acetylation on sialic acid were 7.28, 4.21, and 0.66 at the intact protein level, respectively. For O-glycosylation site S126, O-glycosylation ratios analyzed at the intact protein level and the glycopeptide level were 80.2% and 80.3%, respectively, and two O-glycans were identified, including Core1_S1 and Core1_S2. This study also compared the difference of the glycans and their relative contents in batch-to-batch rhEPO samples. The results proved that the workflow using EAD fragmentation in LC-MS method could be effectively applied for characterizing the glycosylation analysis of rhEPO samples and batch-to-batch consistency analysis, which would help to reasonably guide the optimization of rhEPO production process.

Titer and charge-based heterogeneity multiattribute monitoring of mAbs in cell culture harvest using 2D ProA CEX MS

Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60–90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 μg to 160 μg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.

A simple, rapid, and green method for determination of three cholic acids in bio-transformed Jindanfen by HPLC-MS

AbstractA simple, rapid, and green high-performance liquid chromatography-mass spectrometry (HPLC-MS) method was developed for determination of tauroursodeoxycholic acid (TUDCA), taurocholic acid (TCA), and taurochenodeoxycholic acid (TCDCA) in bio-transformed Jindanfen (BTJDF), which is obtained from chicken bile through a bioconversion process. The sample was prepared using water. The HPLC separation was operated on a poroshell 120 EC-C18 column with a 2.0 min gradient elution procedure. Detection was performed on a single quadrupole mass spectrometer in negative mode with selected ion monitoring mode (SIM). This developed HPLC-MS method presented good linearity (r &gt; 0.997) and sensitivity (limit of quantification, 30.0–80.0 pg) for three analytes. The relative standard deviations (RSDs) for precision, repeatability, and stability were all below 3.00%. The matrix effects and average recoveries of three analytes were 91.2–97.9% (RSDs &lt; 1.50%) and 95.4–103% (RSDs &lt; 3.00%), respectively. The average contents of TUDCA, TCA, and TCDCA in ten batches of samples were 33.8, 13.2, and 20.5%, respectively. Finally, the greenness of the developed method was validated by Analytical Eco-Scale and Complex-GAPI. The developed method was proved to be an eco-friendly, effective, and reliable approach for assaying the three cholic acids in BTJDF, which is help to improve the quality evaluation level of the BTJDF industry.

IDENTIFICATION AND QUANTIFICATION OF FLAVONOIDS IN MEDICINAL PLANTS GROWING IN THE REPUBLIC OF UZBEKISTAN

Introduction. In recent years, the popularity of herbal medicine has been increasing. One of the most numerous classes of biologically active substances of plant origin are flavonoids, which have a wide range of biological activities: anticarcinogenic, antioxidant, antimicrobial, neuroprotective, immunomodulatory, hepatoprotective effects, and also capable of stimulating the damaged body tissues regeneration processes. The relevant area of the research is the search for medicinal plants with a high content of flavonoids for the development of herbal remedies with wound-healing activity. This problem can be solved through the use of plant raw materials from the Republic of Uzbekistan, which have a high content and a wide range of biologically active substances, thanks to the sharply continental climate of the country with consistently hot summers and increased levels of ultraviolet radiation. The purpose of the research is to select medicinal plant raw materials as a source of flavonoids for the development of wound healing agents. Material and methods. The object of the study was medicinal plant raw materials harvested on the territory of the Republic of Uzbekistan: oregano herb, sage leaves, St. John's wort herb, sandy everlasting flowers, bur beggar-ticks herb. Identification and quantification of flavonoids in the studied samples of medicinal plants was carried out using thin-layer chromatography, absorption spectrophotometry in the visible region using reaction with aluminum chloride and high-performance liquid chromatography-mass spectrometry. Results. It has been established that the content of flavonoids in the herb of oregano, leaves of medicinal sage, St. John's wort herb, flowers of sandy everlasting, harvested in the Republic of Uzbekistan, is significantly higher than in medicinal plant materials harvested in the territory of the Republic of Belarus and the Russian Federation. Using the method of thin-layer chromatography and high-performance liquid chromatography-mass spectrometry, kaempferol-3-β-D-glucopyranoside was identified in the extract of sandy everlasting flowers, and isoquercitrin was identified in the extract of St. John's wort herb. Conclusions. As a result of the research, sandy everlasting and St. John's wort, growing on the territory of the Republic of Uzbekistan, were selected as the sources of flavonoids (glycoside kaempferol and isoquercitrin) with regenerating properties for the development of wound healing agents.

Impact of Social Restrictions During COVID-19 on the Aquatic Levels of Antimicrobials and Other Drugs in Delhi.

The relative contribution of factors responsible for the environmental exposure of active pharmaceutical ingredients (APIs) is of interest for appropriate remedial measures. This study was carried out to evaluate the post-lockdown levels of APIs in water resources, in comparison to our previously published study from 2016. The environmental levels of 28 drugs from different classes were analyzed in surface water (Yamuna River), aquifers, and leachate samples collected from 26 locations in Delhi-NCR using the previously validated liquid chromatography-mass spectrometry (LC-MS/MS) methods. In addition, the prevalence of antimicrobial resistance in coliforms isolated from targeted surface water samples was also studied. This study revealed that more than 90% of APIs, including antibiotics, decreased drastically in both surface water and aquifers compared to our previous data. Selected samples subjected to antimicrobial resistance (AMR) analysis revealed the presence of cephalosporin-resistant coliform bacteria. Tracing cephalosporins in the surface and drain water samples revealed the presence of ceftriaxone in the drain and water samples from Yamuna River. Higher levels of ceftriaxone in landfill leachate were also found, which werefound to be associated with coliform resistanceand indicatethe un-segregated disposal of medical waste into landfills. Social restrictions enforced due to COVID-19 resulted in a drastic decrease in antimicrobials and other APIs in aquatic water resources. Increased ceftriaxone and cephalosporin resistance was seen in coliform from surface water and drain, indicating the possibility of hospital waste and treatment-related drugs entering Yamuna River. Enforcement of the regulations for the safe disposal of antibiotics at hospitals and preliminary disinfection of hospital sewage before its inflow into common drains might help minimize the spread of antibiotic resistance in the environment.

Aptamer-free upconversion nanoparticle/silk biosensor system for low-cost and highly sensitive detection of antibiotic residues

The detection of antibiotics is crucial for safeguarding the environment, ensuring food safety, and promoting human health. However, developing a rapid, convenient, low-cost, and sensitive method for antibiotic detection presents significant challenges. Herein, an aptamer-free biosensor was successfully constructed using upconversion nanoparticles (UCNPs) coated with silk fibroin (SF), based on Förster resonance energy transfer (FRET) and the charge-transfer effect, for detecting roxithromycin (RXM). A synergistic FRET efficiency was achieved by utilizing alizarin red and RXM complexes as energy acceptors, with UCNP as the energy donor, and immobilizing an ultrathin SF protein corona within 10 nm. The biosensor detects RXM in deionized water with high sensitivity primarily through monolayer adsorption, with a detection range of 1.0 nM–141.6 nM and a detection limit as low as 0.68 nM. The performance of this biosensor was compared with the ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for detecting antibiotics in river water separately and a strong correlation between the two methods was observed. The biosensor exhibited long-term stability in aqueous solutions (up to 60 d) with no attenuation of fluorescence intensity. Furthermore, the biosensor's applicability extended to the highly sensitive detection of other antibiotics, such as azithromycin. This study introduces a low-cost, eco-friendly, and highly sensitive method for antibiotic detection, with broad potential for future applications in environmental, healthcare, and food-related fields.

LipiDex 2 Integrates MSn Tree-Based Fragmentation Methods and Quality Control Modules to Improve Discovery Lipidomics.

As lipidomics experiments increase in scale and complexity, data processing tools must support workflows for new liquid chromatography-mass spectrometry (LC-MS) methods while simultaneously supporting quality controls to maximize the confidence in lipid identifications. LipiDex 2 improves lipidomics data processing algorithms from LipiDex 1 and introduces new tools for spectral matching and peak annotation functions, with improvements in speed and user-friendliness. In silico spectral library generation now supports tandem mass spectral (MSn) tree-based fragmentation methods, and the LipiDex 2 workflow fully integrates the fragmentation logic into the data processing steps to enable lipid identification at the appropriate level of structural resolution. Finally, LipiDex 2 features new modules for automated quality control checks that also allow users to visualize data quality in a data dashboard user interface.

Targeted screening of multiple anti-inflammatory components from Chrysanthemi indici Flos by ligand fishing with affinity UF-LC/MS.

Chrysanthemi indic Flos (CIF) has been commonly consumed for the treatment of inflammation and related skin diseases. However, the potential bioactive components responsible for its anti-inflammatory and sensitive skin (SS) improvement activities, and the correlated mechanisms of action still remain unknown. In this work, it was firstly found that the CIF extract (CIFE) displayed arrestive free radical scavenging activity on DPPH and ABTS radicals, with no significant difference with positive control Trolox (p > 0.05). Then, compared to the negative group, CIFE markedly decreased the productions of the pro-inflammatory cytokines (IL-1β, IL-6, PEG2, TNF-α, IFN-γ, NO) in LPS induced RAW264.7 cells in a dose-dependent manner (p < 0.01). Besides, CIFE strongly inhibited the COX-2 and hyaluronidase (HAase) with the IC50 values of 1.06 ± 0.01μg/mL and 12.22 ± 0.39μg/mL, indicating higher inhibitory effect than positive control of aspirin of 6.33 ± 0.05μg/mL (p < 0.01), and comparable inhibitory effect with indometacin of 0.60 ± 0.03μg/mL, and ascorbic acid of 11.03 ± 0.41μg/mL (p > 0.05), respectively. Furthermore, kinetic assays with Lineweaver-Burk plot (Michaelis Menten equation) suggested that CIFE reversibly inhibited the COX-2 and HAase, with a mixed characteristics of competitive and non-competitive inhibition. Thereafter, multi-target affinity ultrafiltration liquid chromatography-mass spectrometry (UF-LC/MS) method was employed to fast fish out the potential COX-2 and HAase in CIFE. Herein, 13 components showed various affinity binding degrees to the COX-2 and HAase, while those components with relative binding affinity (RBA) value higher than 3.0, such as linarin and chlorogenic acid isomers, were deemed to be the most bioactive components for the anti-inflammatory and SS improvement activities of CIFE. Finally, the interaction mechanism, including binding energy, inhibition constant, docking sites, and the key amino acids involved in hydrogen bonds between the potential ligands and COX-2/HAase were simulated and confirmed with the molecule docking analysis. In summary, this study showcased the prominent anti-inflammatory and SS improvement activities of CIF, which would provide further insights on this functional medicinal plant to be a natural anti-SS remedy.

Highly sensitive liquid chromatography-mass spectrometry method for the quantitative analysis of mometasone furoate in human plasma: Method validation and application to clinical pharmacokinetic studies.

We report the development and the validation of a sensitive liquid chromatography-mass spectrometry (LC-MS/MS) method for mometasone furoate (MF) analysis in human plasma. Plasma samples were processed through liquid-liquid extraction and analyzed using LC-MS/MS operating in positive mode using multiple reaction monitoring of transitions m/z 520.9→ 355.0 and m/z 525.8→ 355.0 for MF and the internal standard (IS), respectively. Separation was achieved at 1.0mL/min on a C18 column using a gradient elution of mobile phase of 0.05% ammonia in water (phase A) and acetonitrile (phase B). The assay range was 0.250-100 pg/mL and proved to be accurate and precise MF. Normalized recoveries were consistent and reproducible with a coefficient of variation (CV%) value of 6.0. The CV (%) of the IS normalized matrix factor was not observed in normal, lipemic, and hemolyzed plasmas. Dilutions of 1:10 were accurately quantified. A cycle of three freeze and thaw and stabilities at room temperature and on the autosampler were demonstrated. In addition, MF in the presence of indacaterol and glycopyrronium was proven to be stable at -70°C for at least 157 days. The present method was successfully applied to quantify MF in patients receiving MF, indacaterol, and glycopyrronium as a fixed-dose combination.