Chromatography-electrospray Ionization-tandem Mass Spectrometry Research Articles

Comprehensive analysis of Moringa oleifera leaves’ antioxidant properties in ovine cheese

This study aimed to enhance “Pecorino” type ovine cheese by adding Moringa oleifera leaves powder (MOLP). Cheese-making trials, conducted at industrial level, used raw ewes' milk and two selected Lactococcus lactis strains. The experimental plan included a control production (CTR), and two experimental productions with 1% or 2% MOLP addition (1-MOLP and 2-MOLP, respectively). MOLP did not hinder starters development, which reached about 8.0 Log CFU/g in 2-month ripened cheeses. Illumina results highlighted lactococci dominance in all trials [45.98%–62.48% of relative abundance (RA)]. Physicochemical analysis showed that MOLP-enriched cheeses had higher protein content and lower secondary lipid oxidation. The addition of MOLP increased total phenolic compounds in cheese, reaching 3.64 mg GAE/g in the 2-MOLP sample. MOLP-enriched cheeses showed significantly higher radical scavenging activity than CTR production (p < 0.0001). Ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) revealed increased levels of chlorogenic acid, protocatechuic acid, caffeic acid, and ferulic acid due to MOLP enrichment. In the presence of MOLP, cheese volatile organic compounds were affected by compounds like 2-octanone, 3-hexen-2-one, heptane, nonanol, and linalool. 1-MOLP cheese was comparable to CTR production in overall satisfaction (sensory evaluation). Including MOLP in cheese production offers exciting opportunities for functional Sicilian ewes’ milk products.

Mercaptans in malodorants break disulfide bridges in human serum albumin and form adducts suitable as biomarkers of exposure in vitro.

Malodorants comprise notoriously smelling mercaptans and might be applied for crowd control. Because exposure to malodorants may lead to irritation of the respiratory system, choking, and coma, bioanalytical verification of poisoning might be required in a medical and forensic context. We herein present the detection and identification of novel biomarkers of exposure to ethyl mercaptan, n-butyl mercaptan, tert-butyl mercaptan, and iso-amyl mercaptan. These alkyl thiol compounds were found to form disulfide adducts in human serum albumin (HSA) in plasma in vitro with the only non-disulfide-bridged Cys34 residue and with other residues being part of the disulfide-bridged pattern in HSA. After proteinase K-catalyzed proteolysis, adducts of all mercaptans were detected simultaneously as the tripeptide Cys34*ProPhe and the dipeptides Cys369*Tyr, ValCys316*, and Cysx*Ala (x denominates either Positions 91, 200, 253, 361, and/or 448) by a sensitive micro-liquid chromatography-electrospray ionization tandem mass spectrometry (μLC-ESI MS/MS) method working in the scheduled multiple reaction monitoring (sMRM) mode. Time- and concentration-dependent adduct formations while exposure and proteolysis were investigated and the suitability of adducts as biomarkers of exposure was elaborated. Adducts at Cys34 showed the lowest limits of identification (LOIs, 6nM to 1.2μM mercaptan in plasma) and superior stability in plasma at 37°C. Therefore, Cys34*ProPhe appears as the most promising target to prove exposure to mercaptans at least in vitro.

Characterization of the covalent binding of cyanidin-3-glucoside to bovine serum albumin and its inhibition mechanism for advanced nonenzymatic glycosylation reactions.

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

Simultaneous Detection Method of 11 Respiratory Drug Substances Including Theobromine, Oxymetazoline, etc. in Adulterated Dietary Supplements Using Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry and Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry Analysis

Abstract Recently, the demand for respiratory disease-related products has surged due to the influence of coronavirus disease 2019, prompting warnings about illegal dietary supplements containing unauthorized substances. Additionally, adulterated dietary supplements are continuously detected in open markets, posing significant public health safety problem. In this study, we developed and validated an analytical method for 11 respiratory drug substances using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) and proposed optimal conditions for LC–quadrupole time-of-flight MS (LC–QTOF-MS) to determine the fragmentation patterns of each substance. This method underwent thorough validation considering specificity, linearity, limits of detection and quantification, accuracy, precision, matrix effect, stability, etc. All results met international guidelines. These validated methods were applied to 52 dietary supplements advertised for treating respiratory diseases and enhancing respiratory function, among which one sample was found to contain 313.7 mg/g of theobromine. This determination was made by comparing the product ion ratios with the standards and subsequent quantification. To re-confirm the detected substances, their fragmentation patterns were compared with those of the standards using LC–QTOF-MS. In conclusion, the mass-based information, coupled with the LC–ESI–MS/MS method development, can be successfully applied to rapidly identify 11 respiratory drug substances in illegal dietary supplements used for respiratory disease treatment. The developed simultaneous detection method contributes to public health and safety improvements.

Effect of saponins in Panax notoginseng (Burkill) F. H. Chen on the steroid hormone levels in the chronic unpredictable mild stress model of depression in rats

Recent research has indicated that Panax notoginseng saponins (PNS) extracted from the radix of Panax notoginseng (Burkill) F. H. Chen exert antidepressant effects. This study aimed to assess the antidepressive effects of ginsenoside Rg1 and PNS in a depression model induced by chronic unpredictable mild stress (CUMS). Over a period of three weeks, rats were administered ginsenoside Rg1 at a dose of 30 mg/kg and PNS at dosages ranging from 100 to 200 mg/kg body weight per day. To assess how ginsenoside Rg1 and PNS influence depression-like behaviours in rats, various assessments were conducted, including coat state evaluation, forced swim test, and elevated plus maze test. The levels of cortisol and testosterone in serum samples were analysed using the liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) method. LC-ESI-MS/MS method provides precise and accurate results. The lower limit of quantification values for cortisol and testosterone were determined as 100 and 2 pg/mL, respectively. Our data demonstrated that both ginsenoside Rg1 and PNS significantly reversed depression-like behaviour in rats by improving coat condition, reducing immobility time in the forced swim test, and increasing time spent in the open arms of the elevated plus maze test. Furthermore, ginsenoside Rg1 and PNS exhibited a regulatory effect on cortisol and testosterone levels in plasma. These findings suggest that ginsenoside Rg1 and PNS may be potential antidepressants in clinical treatment.

Tandem mass spectrometry imaging to assess illudin S distributions in Omphalotus illudens mushrooms

Poisoning metabolites within mushrooms typically exert varying physiological and biological effects; however, their distributions within mushrooms are seldom investigated. This study focused on imaging illudin S, a representative poisoning metabolite within Omphalotus illudens mushroom, using desorption electrospray ionization–tandem mass spectrometry (DESI–MS/MS) coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Initially, LC–ESI–MS/MS conditions were established for the selective detection of illudin S (m/z 247→159, positive mode) from extracted mushroom. Based on this MS/MS condition, each mushroom was divided into nine sections, and each section was subjected to specific DESI–MS/MS mode imaging (pixel size: 200 × 200 μm). The results revealed high concentrations of illudin S in the stalks of the mushrooms growing on trees. Further, re-evaluations using the LC–MS/MS assay revealed a seven-fold difference in the illudin S between the stalk and other regions. This study marks the first attempt at assessing toxic metabolite distributions within poisonous mushrooms, thus offering valuable insights including the efficient utilization of illudin analogs in biological applications.

Quantitative analysis of erythromycin, its major metabolite and clarithromycin in chicken tissues and eggs via QuEChERS extraction coupled with ultrahigh-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and novel method involving ultrahigh-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UHPLC-ESI–MS/MS) was developed to simultaneously detect erythromycin, its major metabolite and clarithromycin in chicken tissues (muscle, liver and kidney) and eggs (whole egg, albumen and yolk). Samples were extracted using acetonitrile–water (80:20, v/v), and a Cleanert MAS-Q cartridge was used to perform quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification. The average recoveries were 87.78–104.22 %, and the corresponding intraday and interday relative standard deviations were less than 7.10 %. The decision limits and detection capabilities of the chicken tissues and eggs were 2.15–105.21 μg/kg and 2.26–110.42 μg/kg, respectively. For chicken tissues and eggs, the limits of detection and limits of quantification were 0.5 μg/kg and 2.0 μg/kg, respectively. The proposed method was successfully employed to analyse real samples, demonstrating its applicability.

A Reliable Quantification of Cholesterol and 25-Hydroxycholesterol in Liposomal Adjuvant Formulation by Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Cholesterol, as one of the major components of liposomes, plays a critical role in modulating membrane bilayer permeability, fluidity, and structural stability. Controlling these quality attributes is essential to maintaining the efficacy and fitness of the liposomes in various applications. However, during the manufacture and storage of liposomes, cholesterol has a propensity to undergo oxidative degradation. Hence, an analytical tool that is capable of determining not only the identity and quantity of cholesterol but also its associated degradants is a prerequisite to effective process control and product quality and safety assessments. In this view, a new liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method with parallel reaction monitoring (PRM) was developed and qualified to accurately quantify cholesterol and monitor the formation of 25-hydroxycholesterol degradant in liposomal drug formulations without the use of an isotopic internal standard (IS). The method was qualified according to the FDA Quality Guidance for Industry: Q2(R1). Study results showed that the method presents good specificity for cholesterol and 25-hydroxycholesterol detection in the liposomal matrix, good sensitivity characterized by LOD/LOQ in the nanomolar range, and accuracy within the range of 80 to 120%. The described method enables accurate evaluation of in-process and product release samples of Army Liposome Formulation with QS21 (ALFQ).

Untargeted Metabolomic Profiling of Fructus Chebulae and Fructus Terminaliae Billericae

This study aims to identify the differences in metabolites between Fructus Chebulae (FC) and Fructus Terminaliae Billericae (FTB). Untargeted metabolomics was used to analyze differentially expressed metabolites (DEMs) with ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS). A grand total of 558 metabolites were detected, with 155 in positive ion mode and 403 in negative ion mode. Further differential analysis yielded 110 and 87 significantly different metabolites, which were mainly polyphenols, flavonoids, terpenoids, and alkaloids. Analysis of KEGG data showed that differentially expressed metabolites (DEMs) in both positive and negative ion modes were found to be enriched in 5 and 18 metabolic pathways, respectively, with metabolic pathways being the most enriched among them. In sum, this study reveals the differential metabolic profiles of FC and FTB and provides support for their further applications in traditional Chinese medicine.

Molecular identification and management of mycotoxigenic fungi in stored corn Grains

AbstractMycotoxin-producing molds which considered as common maize grains contaminants are the genera Fusarium, Aspergillus and Penicillium. There are natural and safe ways to protect grains from mold contamination as the use of essential oils and chemical treatments. A total number of 25 samples were used to study the natural frequency in five governorates in Egypt, Molecular identification indicated that the most frequent fungi were Fusarium verticillioides, Aspergillus niger, Talaromyces verruculosus, Aspergillus flavus and Aspergillus terreus. The in vitro studies have been done to determine mycelial growth and spore germination inhibition of the two A. flavus; isolated and reference isolates. Thyme and acetic acid were tested in direct contact assay to study their effects on mycelial growth. Treatments showed significant impact on mycelial growth and spore germination inhibition of both A. flavus isolates. In the postharvest application treatments: as vapour and carrier contact assay, Thyme and Acetic acid were tested to determine their influence on growth and aflatoxin production in A. flavus isolates by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Results indicated that both treatments were effective in inhibition of aflatoxin production in both vapour and carrier assays as they succeeded in reducing AFB1 while they inhibited completely the production of AFB2. The extent of the inhibition of aflatoxin production was dependent on the concentration and storage duration of treatments applied.

Anti-Cholinergic Effects of the Phenolic Extract from the Astragalus crenatus Plant: A Computational and Network Pharmacology Study.

Investigations into cholinesterase inhibition have received attention from researchers in recent years for the treatment of Alzheimer's disease. Cholinesterase enzymes, namely, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), hold pivotal significance in Alzheimer's disease (AD) treatment. In this study, we utilized the ethanolic extract of Astragalus crenatus followed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to separate and identify at least 21 compounds in the extract. Rosmarinic acid exhibited the highest concentration (96.675 ± 1.3 mg/g extract), succeeded by hesperidin (79.613 ± 1.2 mg/g extract), hesperetin (75.102 ± 1.4 mg/g extract), rutin (68.156 ± 1.6 mg/g extract), chlorogenic acid (67.645 ± 1.5 mg/g extract), fisetin (66.647 ± 2.3 mg/g extract), and hyperoside (63.173 ± 1.5 mg/g extract). A. crenatus extract efficiently inhibited both AChE and BChE activities in a dosage-dependent manner. Molecular docking was employed to scrutinize the anticholinesterase mechanisms of the identified phytocompounds. Notably, a network pharmacology analysis was executed for the most efficacious compound. Based on binding energies, hesperidin emerged as the most potent inhibitor against both AChE and BChE, exhibiting scores of -10.5 Kcal/mol and -9.8 Kcal/mol, respectively. Due to its dual inhibition of AChE and BChE activities, hesperidin from Astragalus crenatus holds promise for the development of novel therapeutics aimed at neurological disorders, particularly AD.

Optimized Conditions for the Extraction of Phenolic Compounds from Aeginetia indica L. and Its Potential Biological Applications.

Aeginetia indica L., a parasitic root in the Orobanchaceae family, is used as a food colorant in traditional Thai desserts. However, scant information is available on its food applications as well as medicinal properties, while overharvesting by the local people has severely depleted wild plant populations. This research, thus, aimed to extract optimized total phenolic content (TPC) in varying extraction conditions using response surface methodology (RSM) and the Box-Behnken design (BBD). Results indicated that an extraction temperature of 90 °C, 80% (v/v) aqueous ethanol, and 0.5% (w/v) solid-to-liquid ratio yielded the highest TPC at 129.39 mg gallic acid equivalent (GAE)/g dry weight (DW). Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) identified the predominant phenolics as apigenin (109.06 mg/100 g extract) and luteolin (35.32 mg/100 g extract) with trace amounts of naringenin and rutin. Under the optimal extraction condition, the plant extract exhibited antioxidant activities of 5620.58 and 641.52 µmol Trolox equivalent (TE)/g DW determined by oxygen radical absorbance capacity (ORAC) and ferric ion reducing antioxidant power (FRAP) assay, while the scavenging capacity of total radicals at 50% (SC50) was determined to be 135.50 µg/mL using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The plant extract also exhibited inhibitory activities against the key enzymes relevant to type II diabetes, obesity, and Alzheimer's disease, suggesting the potential for medicinal applications.

Signal interference between drugs and metabolites in LC-ESI-MS quantitative analysis and its evaluation strategy

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS) is a widely utilized technique for in vivo pharmaceutical analysis. Ionization interference within electrospray ion source, occurring between drugs and metabolites, can lead to signal variations, potentially compromising quantitative accuracy. Currently, method validation often overlooks this type of signal interference, which may result in systematic errors in quantitative results without matrix-matched calibration. In this study, we conducted an investigation using ten different groups of drugs and their corresponding metabolites across three LC-ESI-MS systems to assess the prevalence of signal interference. Such interferences can potentially cause or enhance nonlinearity in the calibration curves of drugs and metabolites, thereby altering the relationship between analyte response and concentration for quantification. Finally, we established an evaluation scheme through a step-by-step dilution assay and employed three resolution methods: chromatographic separation, dilution, and stable labeled isotope internal standards correction. The above strategies were integrated into the method establishment process to improve quantitative accuracy.

Breast milk concentrations of acetaminophen and diclofenac - unexpectedly high mammary transfer of the general-purpose drug acetaminophen

BackgroundBreastfeeding is considered to be the most effective way of ensuring the health and survival of newborns. However, mammary transfer of drugs administered to mothers to breastfeeding infants remains a pressing concern. Acetaminophen and diclofenac sodium are widely prescribed analgesics for postpartum pain relief, but there have been few recent reports on the mammary transfer of these drugs, despite advances in analytic techniques.MethodsWe conducted a study on 20 postpartum mothers from August 2019–March 2020. Blood and milk samples from participants were analyzed using liquid chromatography-electrospray ionization tandem mass spectrometry within 24 hours after oral administration of acetaminophen and diclofenac sodium. The area under the concentration-time curve (AUC) was calculated from the concentration curve obtained by a naive pooled-data approach.ResultsFor acetaminophen, AUC was 36,053 ng/mL.h and 37,768 ng/mL.h in plasma and breast milk, respectively, with a milk-to-plasma drug concentration ratio of 1.048. For diclofenac, the AUC was 0.227 ng/mL.h and 0.021 ng/mL.h, in plasma and breast milk, respectively, with a milk-to-plasma drug concentration ratio of 0.093.ConclusionsWhile diclofenac sodium showed low mammary transfer, acetaminophen showed a relatively high milk-to-plasma drug concentration ratio. Given recent studies suggesting potential connections between acetaminophen use during pregnancy and risks to developmental prognosis in children, we believe that adequate information regarding the fact that acetaminophen is easily transferred to breast milk should be provided to mothers.

The phosphylated butyrylcholinesterase-derived tetrapeptide GlyGluSerAla proves exposure to organophosphorus agents with enantioselectivity.

We herein present for the first time the phosphylated (*) tetrapeptide (TP)-adduct GlyGluSer198*Ala generated from butyrylcholinesterase (BChE) with proteinaseK excellently suited for the verification of exposure to toxic organophosphorus nerve agents (OPNA). Verification requires bioanalytical methods mandatory for toxicological and legal reasons. OPNA react with BChE by phosphonylation of the active site serine residue (Ser198) forming one of the major target protein adducts for verification. After its enzymatic cleavage with pepsin, the nonapeptide (NP) PheGlyGluSer*AlaGlyAlaAlaSer is typically produced as biomarker. Usually OPNA occur as racemic mixtures of phosphonic acid derivatives with the stereocenter at the phosphorus atom, e.g. (±)-VX. Both enantiomers react with BChE, but the adducted NP does not allow their chromatographic distinction. In contrast, the herein introduced TP-adducts appeared as two peaks when using a stationary reversed phase (1.8µm) in micro-liquid chromatography-electrospray ionisation tandem-mass spectrometry (µLC-ESI MS/MS) analysis. These two peaks represent diastereomers of the (+)- and (-)-OPNA adducted to the peptide that comprises chiral L-amino acids exclusively. Concentration- and time-dependent effects of adduct formation with (±)-VX and its pure enantiomers (+)- and (-)-VX as well as with (±)-cyclosarin (GF) were investigated in detail characterising enantioselective adduct formation, stability, ageing and spontaneous reactivation. The method was also successfully applied to samples from a real case of pesticide poisoning as well as to samples of biomedical proficiency tests provided by the Organisation for the Prohibition of Chemical Weapons.

Abstract A068: Proteomic analysis of keratin 17 positive cells from laser-capture microdissected pancreatic ductal adenocarcinoma

Abstract Introduction. Although patients with pancreatic ductal adenocarcinoma (PDAC) have one of the lowest survival of all carcinomas, there is considerable variation in survival and disease progression, even when accounting for factors such as age and pathologic stage. Our group identified keratin 17 (K17) as a negative prognostic and predictive biomarker in PDAC. Patients with high K17 expression in their tumors have shorter overall survival as well as decreased response to chemotherapeutic agents, including gemcitabine. K17 has also emerged as an established biomarker of the basal molecular subtype of PDAC. In this study, we aim to identify proteins which correlate, directly or indirectly, with K17 expression via a combined approach using immunohistochemistry (IHC) followed by laser capture microdissection (LCM) and mass spectrometry. Methods. We selected 5 PDAC cases of low K17 expression and 5 PDAC cases of high K17 expression from the archival collections from the Stony Brook Cancer Center Biobank. A modified indirect immunoperoxidase approach was performed on sections mounted on LCM-compatible membrane slides. Carcinoma cells that were either K17 positive or negative by IHC were precisely dissected using LMD7 microscope (Leica) from 10-µM thick sections, followed by a modified tissue digestion and protein extraction protocol. Collision-induced fragment mass information and peptide abundance values were obtained by a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS). Spectral cutoffs were analyzed at both &amp;lt;5% and 1% false discovery rates. The human UniProt dataset was used for data alignment. Results. Mass spectrometry yielded over 1600 detected proteins with approximately 50 of which correlated strongly with K17 expression. Our preliminary results show that keratin 5, a known marker of basal-like PDAC, is positively correlated with keratin 17. Genes whose increased expression contribute to the Moffitt classification of basal molecular subtype of PDAC, such as S100A2, keratin 6A and keratin 7, also showed positive correlation with K17 in our study. The remainder of proteins associated with K17 expression include those involved with cell adhesion, cell-extracellular matrix interaction and smooth muscle contraction pathways, with specific types and classes being identified, pending in-depth analysis. Proteins with inverse relationship to K17 expression included those involved in intracellular organelle trafficking and apoptosis pathways. Conclusion. Our findings demonstrate the utility of mass spectrometry-based spatial proteomic analysis of microdissected cell populations from formalin-fixed, paraffin embedded samples. These methods have the potential to uncover complementary biomarkers to K17 that could enhance prognostic accuracy or serve as therapeutic targets and provide mechanistic insight into the mechanisms through which K17 drives tumor aggression. Citation Format: Ji Dong K. Bai, John D. Haley, Natalia Marchenko, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. Proteomic analysis of keratin 17 positive cells from laser-capture microdissected pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A068.

Ethanolysis of nitrogen mustards: A novel strategy for nitrogen mustard identification in environmental matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Nitrogen mustards (NMs) are blistering chemical warfare agents. The ability to detect NMs in environmental samples is very important for obtaining forensic evidence. The most common analytical techniques for NM detection are gas chromatography-mass spectrometry, which detects NMs in their intact form but is disadvantaged by high limits of detection (LODs), and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) of their hydrolysis products, which do not provide robust evidence to support NM use. We developed a novel approach to detect and identify NMs using LC/ESI-MS/MS after chemical derivatization. The method is based on ethoxide-promoted ethanolysis prior to analysis. The effects of reaction time, temperature, ethoxide concentration and chromatography behavior were studied and optimized. In the developed procedure, 0.1% (v/v) sodium ethoxide solution is added to the NMs in ethanol and agitated for 2h at 50°C, followed by LC/ESI-MS/MS, without any other pretreatment. The ethanolysis reaction efficiencies were evaluated in ethanolic extracts from soil, asphalt, and ethanol contaminated with 0.5% (v/v) diesel fortified with NMs at a five-point calibration curve. The calibration curves showed good linearity in the range of 0.05-1ng/mL, with an R2 value of 0.99, and were similar to those of LC/MS-grade ethanol, with almost no observable matrix effects. The derivatization products were stable at room temperature, with LODs of 10pg/mL, in all investigated extracts. Through this newly developed strategy, the derivatization of active NMs by ethanolysis was achieved for the first time, and these derivatization products can serve as specific indicators for the use and presence of NMs. The methodology can also verify trace levels of NM chemical warfare agents collected in war or terror scenarios in forensic investigations.

Study on the Underlying Mechanism of Yinhua Gout Granules in the Treatment of Gouty Arthritis by Integrating Transcriptomics and Network Pharmacology.

Yinhua Gout Granules (YGG) is a traditional Chinese medicine preparation with a variety of pharmacological effects, and its clinical efficacy in the treatment of gouty arthritis (GA) has been fully confirmed. However, the pharmacodynamic basis of YGG and its anti-inflammatory mechanism of action in GA are unknown. The objective of this study was to identify the active components and molecular mechanisms of YGG in the treatment of GA. Ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) and network pharmacology were used to identify and predict the potential active ingredients and related signaling pathways. Then, we revealed the anti-GA effects of YGG based on pharmacodynamic experiments in GA rats. Finally, we integrated transcriptomics and network pharmacology to elucidate the potential mechanism of action and verified the putative mechanism by molecular docking, immunohistochemical (IHC) and Western blot. We have identified 10 major active components of YGG that may have anti-GA effects, such as ferulic acid, rutin, luteolin, etc. Using molecular docking, we found that 10 major compounds could bind well to TNF, PTGS2, IL-6, IL1β, NOS2 and PTGS1, and the binding energies were all less than -5 kcal/mol. Animal studies have shown that YGG can improve joint inflammation and inflammatory cell infiltration, reduce serum UA, BUN and Cr levels (p<0.01), and decrease IL-1β, IL-6, TNF-α, COX-2 and PGE2 levels in synovial tissue (p<0.01), which are associated with the pathogenesis of GA. IHC and Western blot results showed that YGG could regulate TLR4/MYD88/NF-κB pathway to inhibit the inflammatory response induced by GA. This study found that YGG could not only improve the disease of GA by inhibiting the production of UA in the body, but also target the regulation of TLR4/MYD88/NF-κB signaling pathway through a variety of active components to achieve effective therapeutic effects on GA.

Association of phthalate exposure with all-cause mortality across renal function status: The U.S. National Health and Nutrition Examination Survey, 2005–2018

BackgroundWide phthalate exposure has been associated with both declines in renal function and an elevated risk of mortality. Whether phthalate-associated risk of premature mortality differs by renal function status remains unclear. MethodsThis study included 9605 adults from the U.S. National Health and Nutrition Examination Survey. Urinary concentrations of 11 phthalate metabolites were assessed using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. According to estimated glomerular filtration rate (eGFR), participants were grouped as having normal or modestly declined renal functions, or chronic kidney disease (CKD). Multivariable Cox regression models estimated all-cause mortality associated with phthalate exposure, overall and by renal function status. ResultsOverall, Mono-n-butyl phthalate (MnBP), Mono-benzyl phthalate (MBzP), Mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and Mono-(2-ethyl-5-carbox-ypentyl) phthalate (MECPP) were associated with an elevated risk of mortality (P-trend across tertile <0.05). Moreover, significant interactions were observed between eGFR and MEHHP, MEOHP, MECPP, DEHP in the whole population (P for interactions <0.05). After stratification by renal function, total Di (2-ethylhexyl) phthalate (DEHP) was additionally found to be associated with mortality risk in the CKD group (HR = 1.12; 95% CI: 1.01, 1.25). Co-exposure to the 11 phthalate metabolites was associated with a higher risk of all-cause mortality in the CKD (HR = 1.47; 95% CI: 1.18, 1.84) and modestly declined renal function group (HR = 1.25; 95% CI: 1.09, 1.44). ConclusionsThe associations between phthalate exposure and risk of all-cause mortality were primarily observed in CKD patients, reinforcing the need for monitoring phthalate exposure in this patient population.

Antioxidant, AChE inhibitory, and anticancer effects of Verbascum thapsus extract.

Verbascum thapsus (VT) is a medicinal plant that is used in folk medicine to treat a variety of ailments. For this study, the biological functions of VT methanol extract were determined in vitro. The plant's methanol extract was created through the maceration process. The phytochemical composition of plant extracts was investigated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The antioxidant capacity of the extract was determined using the DPPH (2,2-diphenyl-1-picrylhydrazil) and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) tests and its cytotoxicity was assessed using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole)) assay on the Caco-2 (human colorectal adenocarcinoma cells), LNCaP (Lymph Node Carcinoma of the Prostate), and HEK293 cell lines (Human embryonic kidney 293 cells) used to model colon, prostate, and non-cancerous cells. VT extract showed low DPPH and ABTS radical scavenging activities compared to standard antioxidants at 30 mg/ml concentration. In addition, it was determined that VT extract inhibited acetylcholinesterase enzyme.