Tandem Mass Spectrometry Research Articles

Study on chemical profiling of bailing capsule and its potential mechanism against thyroiditis based on network pharmacology with molecular docking strategy.

Bailing capsule (BLC), a drug that is clinically administered to modulate the autoimmune system, exhibits promising therapeutic potential in the treatment of thyroiditis. This study elucidates the chemical profile of BLC and its potential therapeutic mechanism in thyroiditis, leveraging network pharmacology and molecular docking techniques. Utilizing ultra-high-performance liquid chromatography coupled with linear trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS), 58 compounds were identified, the majority of which were nucleosides and amino acids. Utilizing the ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC QqQ MS/MS) strategy, 16 representative active components from six batches of BLCs were simultaneously determined. Network pharmacology analysis further revealed that the active components included 5'-adenylate, guanosine, adenosine, cordycepin, inosine, 5'-guanylic acid, and l-lysine. Targets with higher connectivity included AKT1, MAPK3, RAC1, and PIK3CA. The signaling pathways primarily focused on thyroid hormone regulation and the Ras, PI3K/AKT, and MAPK pathways, all of which wereintricately linked to inflammatory immunity and hormonal regulation. Molecular docking analysis corroborated the findings from network pharmacology, revealing that adenosine, guanosine, and cordycepin exhibited strong affinity toward AKT1, MAPK3, PIK3CA, and RAC1. Overall, this study successfully elucidated the material basis and preliminary mechanism underlying BLC's intervention in thyroiditis, thus laying a solid basis for further exploration of its in-depth mechanisms.

Vitamin D and CRP are associated in hospitalized inflammatory bowel disease (IBD) patients in Shanghai.

Patients with inflammatory bowel disease (IBD) are more likely to be confirmed with vitamin D deficiency. However, the association between inflammation and vitamin D remains unclear. The purpose of this study was to evaluate the association between inflammation and vitamin D in hospitalized patients with IBD. All the participants were recruited from one teaching hospital from June 2018 to October 2022. Inflammation was evaluated by serum concentration of C-reactive protein (CRP), using an immunoturbidimetric method at admission. We further divided the participants into five groups based on serum CRP levels: <5, 5-9.9, 10-19.9, 20-39.9, and >40mg/L. Serum 25-hydroxy-vitamin D (25-(OH)-D) was assessed by liquid chromatography tandem mass spectrometry. Addi-tional information, including age, sex, body mass index (BMI), IBD (ulcerative colitis vs. Crohn's disease) subtype, was abstracted from medical records. This study included 1,989 patients with IBD (average age was 39.4 years, 33.8% of them were women, 1,365 CD and 624 UC patients). The median CRP was 5.49 mg/L (range of quartiles: 1.64~19.5 mg/L) and the prevalence of 25-(OH)-D deficiency was 69.8%. CRP was significantly associated with serum level of 25-(OH)-D. The difference in 25-(OH)-D was -4.28 ng/ml (-5.27 ng/ml, -3.31 ng/ml) between two extremist CRP groups after adjustment of potential covariates (age, sex, BMI, type of IBD, dietary type, season, and lymphocyte count). Subgroup analysis in sex, type of IBD, and age, were similar to the main analysis results. There was a negative association between CRP levels and vitamin D in hospitalized patients with IBD.

A Flavonoid Concentrate from Moringa Oleifera Lam. Leaves Extends Exhaustive Swimming Time by Improving Energy Metabolism and Antioxidant Capacity in Mice.

Moringa oleifera Lam. leaves contain various nutrients and bioactive compounds. The present study aimed to assess the anti-fatigue capacity of a flavonoids concentrate purified from M. oleifera Lam. leaves. The total flavonoids in the purified extract were analyzed by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS/MS). The mice were supplemented with purified M. oleifera Lam. leaf flavonoid-rich extract (MLFE) for 14 days. The weight-loaded forced swimming test was used for evaluating exercise endurance. The 90-min non-weight-bearing swimming test was carried out to assess biochemical biomarkers correlated to fatigue and energy metabolism. UPLC-MS/MS analysis identified 83 flavonoids from MLFE. MLFE significantly increased the swimming time by 60%. Serum lactate (9.9 ± 0.9 vs. 8.9 ± 0.7), blood urea nitrogen (BUN) (8.8 ± 0.8 vs. 7.2 ± 0.5), and nonesterified fatty acid (NEFA) (2.4 ± 0.2 vs. 1.7 ± 0.3) were significantly elevated; phosphoenolpyruvate carboxykinase (PEPCK), glucokinase (GCK), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression were significantly downregulated; and heme oxygenase 1 mRNA expression was significantly upregulated in muscle after swimming. MLFE supplement significantly decreased serum lactate (8.0 ± 1.0 vs. 9.9 ± 0.9), BUN (8.6 ± 0.4 vs. 8.9 ± 0.8), and NEFA (2.3 ± 0.4 vs. 2.4 ± 0.2) and increased the protein and mRNA expression of GCK, PEPCK, and Nrf2. The enhancement of glucose metabolism and antioxidant function by MLFE contributes partly to its anti-fatigue action.

Evaluation of the presence of emerging contaminants in wastewater and seawater using automated solid-phase extraction and ultra-high-performance liquid chromatography coupled to tandem mass spectrometry

The presence of emerging contaminants in water constitutes one of the main ways of human exposure to them, which can cause severe effects on health. In this sense, their adequate elimination through appropriate treatments plays a fundamental role, being necessary to monitor the effluents from wastewater treatment plants, as well as the areas close to their discharge. The aim of the present work has been the development and validation of an analytical methodology to determine 15 organic contaminants of emerging concern in wastewater as well as in seawater samples using an automated solid-phase extraction system and an ultra-high-performance liquid chromatography coupled to tandem mass spectrometry instrument. Matrix-matched calibration showed good linearity with determination coefficients ≥ 0.990. Likewise, matrix effect assessment showed a significant signal suppression in almost all the analytes demonstrating the need to consider such effect for a proper quantification of the analytes. Recovery values ranged from 74.7 to 109% with relative standard deviation values ≤ 20.5% for most analytes. Regarding seawater and wastewater samples, 11 of the target analytes were detected in, at least, one sample at concentrations from below limit of quantification of the method to 217 ± 92 ng L-1 in seawater and up to 2.3·103 ± 107 ng L-1 in wastewater samples.

Multiple Oxidative Modifications on Hemoglobin are Elevated in Breast Cancer Patients as Measured by Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

Breast cancer is strongly connected with elevated oxidative stress. Oxidative modifications of hemoglobin can serve as biomarkers for monitoring oxidative stress status in vivo. The structure of hemoglobin modifications derived from malondialdehyde (MDA) in human blood hemoglobin exists as N-propenal and dihydropyridine (DHP). This study reports the simultaneous quantification of eleven modified peptides in hemoglobin derived from MDA and advanced histidine oxidation in 16 breast cancer patients and 16 healthy women using nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry. The results reveal statistically significant increases in the formation of MDA-derived N-propenal and DHP of lysine and advanced oxidation of histidine in hemoglobin of breast cancer patients with the Mann-Whitney U-test p values < 0.0001 and the AUC of ROC between 0.9277 and 1.0. Furthermore, the elevation in modified peptides is significant in patients with early stages of breast cancer. By measuring these oxidative modifications in hemoglobin from a drop of blood, the role of lipid peroxidation and oxidative stress in breast cancer can be assessed using this sensitive assay.

O96 NICD LACTYLATION OF NOTCH SIGNALING IMPROVES MICROVASCULATION RAREFACTION IN HYPERTENSION

Background and Objective: Microvascular rarefaction is a key pathophysiologic basis of hypertension-related cardiovascular disease. Exercise decreases blood pressure and promotes angiogenesis related to inhibition of NOTCH signaling. The activation of the NOTCH pathway depends on the transcriptional regulation of its intracellular active fragment NICD after nuclear translocation. The post-translational modification status of proteins plays a crucial role in the nuclear translocation effect of transcription factors. Lactylation is influenced by the level of lactate in the body. Exercise can promote glycolysis in endothelial cells, and increase intracellular lactate levels. Therefore, we explored whether the beneficial effects of Exercise were mediated by promoting NICD lactylation, inhibiting the NOTCH signaling, and promoting angiogenesis. Methods: To test our hypotheses, WKYs (normotensive rats) and SHRs (spontaneously hypertensive rats) were given exercise for 12 weeks. Blood pressure was determined by the tail-cuff method, and cardiac function was assessed by echocardiography. Using rats’ retina, gastrocnemius, and myocardium detected microvascular density. The expression levels of NOTCH signaling pathway related proteins and angiogenesis related factors were measured by Western Blotting. The level of lactate was measured by using a lactate assay kit. Protein modification omics was used to detect the modification types and levels of NICD in microvascular endothelial cells, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to detect the lactylation sites of NICD in microvascular endothelial cells. Results: Exercise in SHRs resulted in reduced blood pressure and concentric hypertrophy. Exercise promoted angiogenesis and improved microcirculation rarefaction. Exercise also reduced the production of NOTCH pathway related proteins and increased the expression level of angiogenesis related factors. The lactate level increased after exercise, and the lactylation level of NICD increased. Conclusions: In summary, Exercise decreases hypertension and improves cardiac function in SHRs. Our research results indicated that exercise increases lactate production and induces NICD lactylation, thereby inhibiting the NOTCH signaling, upregulating the expression of angiogenic factors, promoting angiogenesis, and lowering blood pressure.

Bioaccessibility of plastic-related compounds from polymeric particles in marine settings: Are microplastics the principal vector of phthalate ester congeners and bisphenol A towards marine vertebrates?

Marine vertebrates are known to ingest significant amounts of microplastics (MPs). Once ingested, MPs might cause gastrointestinal injuries and serve as a path of harmful plastic components, such as phthalate esters (PAEs) and bisphenol A (BPA) in the food chain. However, there is a lack of standardized in-vitro methods capable of simulating fish uptake of chemicals from MPs in the environment as potential vectors of such contaminants. In this work, leaching and in-vitro oral bioaccessibility testing of PAEs and BPA from MPs were conducted batchwise using artificial seawater and gut fluids mimicking gastric, intestinal, and gastrointestinal compartments of marine vertebrates at physiological temperature. The environmental and physiologically relevant extraction tests were applied to medium-density polyethylene (PE) and polyvinyl chloride (PVC) certified reference materials containing eight PAEs of varying hydrophobicity, namely, dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate, benzylbutyl phthalate, diethylhexyl phthalate, di-n-octyl phthalate, diisononyl phthalate and diisodecyl phthalate, and BPA (only in PE) as MP surrogates with realistic analyte concentrations for primary MPs. The analysis of the leachates/gut fluid extracts was performed via dilute-and-shoot by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Only the most hydrophilic compounds, i.e. DMP, DEP and BPA, were found to get released significantly in saline waters, and exhibited the highest oral bioaccessibility rates (34–83 %). Based on our results, a dual-compartment physiologically relevant gastrointestinal test is recommended for appropriate estimation of fish bioaccessibility. The fish daily intakes of DMP, DEP and BPA from MPs, and seawater ingestion as well were estimated using several contamination scenarios (10th percentile as low level, 50th percentile as medium level and 90th percentile as high level contamination) based on probabilistic distributions and cumulative probability curves of measured environmental concentrations of (i) MP in seawater throughout the world, (ii) DMP, DEP and BPA in beached MP and those sampled in the open ocean (including both incurred and adsorbed contaminants), and (iii) DMP, DEP and BPA in seawater as reported in recent literature. Under a medium-level concentration scenario (50th percentile) in marine settings, and taking the gastrointestinal bioaccessibility factor into account, the daily intake of DMP, DEP and BPA from MPs accounted for a mere 0.02 % of the waterborne contribution. Hence, the ingestion of MPs should not be considered the primary route of fish exposure to BPA and the most polar PAEs in marine environments. However, more studies on the local and global scales using the continuously available data in the literature for mass concentrations of MP and additives in marine settings are needed for further confirmation of our findings.

Identification of bile acids in snake bile by hydrogen/deuterium exchange mass spectrometry and quantitative structure-retention relationship analysis

Natural bile acids, a class of steroids with a valeric acid side chain at the C-17 position, present significant challenges in separation and analysis due to structural similarities, isomerism, and large polarity differences. Therefore, advanced analytical methods are essential for the accurate identification and quantification of bile acids. This study conducted a comprehensive qualitative analysis of bile acids by integrating liquid chromatography-tandem mass spectrometry (LC-MS/MS), hydrogen-deuterium exchange tandem mass spectrometry (HDX-MS/MS), and quantitative structure-retention relationship (QSRR) methods. Firstly, LC-MS/MS conditions were optimized to enhance chromatographic separation and improve the reliability of characteristic fragment ions. MS/MS fragmentation rules for bile acids were derived from the mass spectral data of bile acid standards and validated through HDX-MS/MS experiments. Secondly, potential bile acids in snake bile were identified based on these validated fragmentation rules, and a QSRR model was established to predict the retention times of the proposed structures. Thirdly, HDX-MS/MS was applied to assist in identifying bile acid isomers. Finally, a total of 150 bile acids, including 11 free bile acids (free BA), 5 glyco-bile acids (GBA) and 134 tauro-bile acids (TBA), were detected in snake bile. Thirteen bile acids were accurately characterized by comparing their retention time and MS/MS spectra with standards. Forty-nine bile acids were reasonably annotated using the QSRR model and HDX-MS/MS. This study is notable for being the first to utilize the QSRR and HDX-MS/MS techniques for the annotation of bile acids in snake bile, providing a robust framework for the structural elucidation of these compounds.

Nonalcoholic steatohepatitis increases plasma retention of sorafenib-glucuronide in a mouse model by altering hepatocyte hopping

Hepatocyte hopping is the hepatocyte-to-sinusoid-to-hepatocyte shuttling that increases the efficiency of hepatic elimination of xenobiotics. This phenomenon is mediated via efflux of hepatic metabolites by Mrp3 and reuptake by Oatp transporters in sequential hepatocytes until eventual biliary efflux by Mrp2. Sorafenib-glucuronide (SFB-G), the major metabolite of sorafenib (SFB), undergoes hepatocyte hopping, leading to efficient biliary elimination. Nonalcoholic steatohepatitis (NASH) alters the functioning of transporters involved in hepatocyte hopping. The purpose of this study was to quantify the effect of NASH on the three drug disposition processes of hepatocyte hopping. Male FVB and C57BL6 wild-type (WT), Oatp1a/1b cluster knockout (O−/−), and Mrp2 knockout (Mrp2−/−) mice were fed a methionine and choline deficient (MCD) diet to induce NASH. Mice were administered 10 mg/kg SFB via oral gavage and concentrations of SFB and SFB-G in plasma quantified using liquid-chromatography tandem mass spectrometry. Compared to WT, plasma area under the concentration-time curve (AUC) of SFB-G increased by 108-fold in the O–/–-C group and by 345-fold in the Mrp2–/–-C group. In the WT-NASH group, up-regulation of Mrp3 and decreased Mrp2 function, along with reduced Oatp uptake, elevated SFB-G AUC by 165-fold. SFB-G AUC in the O–/–-NASH group increased by 108-fold compared to WT-C (3.2-fold compared to O–/–-C). SFB-G AUC in the Mrp2–/– -NASH group increased by 450-fold (1.2-fold compared to Mrp2–/– -C). Taken together, the mislocalization of Mrp2 in NASH is a major contributor to the decrease in SFB-G biliary efflux, but decreased Oatp uptake and enhanced sinusoidal efflux also limit the contribution of downstream hepatocytes, resulting in plasma retention that recapitulates the altered pharmacokinetics observed in human NASH.

Cistus libanotis from Algeria: Phytochemical Analysis by GC/MS, HS-SPME-GC/MS, LC-MS/MS and its anticancer activity

In this paper, Cistus libanotis (Cistus clusii Dunal) from Algeria was investigated. This plant is utilized in numerous industries, mostly in perfumery and, more recently, as a raw material for dietary supplements. For the first time, the present study evaluated the anticancer potential of Algerian C. libanotis by performing a comprehensive phytochemical investigation. The volatile fraction of the fresh plant material of C. libanotis was investigated using headspace solid-phase microextraction (HS-SPME) coupled to GC-MS, and the chemical composition of the essential oil (EO) of C. libanotis was examined using GC-MS technique; additionally, the phenolic compounds were identified and quantified using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A healthy cell line (L929) and two cancer cell lines (CAPAN-1 and dld-1) were used to test the ethanolic extract's cytotoxicity. HS-SPME-GC/MS analysis identified sabinene (25.3%) and α-thujene (23.8%) as principal components. In EO the major ingredients were γ-terpinene (17.4%), camphene (16.13%), α-phellandrene (15.36%), and 4-carene (13.54%). Nevertheless, 24 components were identified by LC-ESI-MS/MS analysis and shikimic acid, luteolin and hesperidin were most abundant. Further, the ethanolic extract of C. libanotis showed a significant cytotoxic effect against dld-1 and CAPAN-1 cell lines, while it was less active against the healthy cell line L929.

Bisphenols in daily clothes from conventional and recycled material: evaluation of dermal exposure to potentially toxic substances

Given the increasing concern about chemical exposure from textiles, our study examines the risks of dermal exposure to bisphenol A (BPA), bisphenol S (BPS), bisphenol B (BPB) and bisphenol F (BPF) from conventional and recycled textiles for adults, aiming to obtain new data, assess exposure, and evaluate the impact of washing on bisphenol levels. A total of 57 textile samples (33 from recycled and 24 from conventional material) were subjected to ultrasound-assisted extraction (UAE) followed by ultra-high performance liquid chromatography with tandem mass spectrometry analysis (UHPLC-MS/MS). The BPA and BPS concentrations varied widely (BPA: < 0.050 to 625 ng/g, BPS: 0.277–2,474 ng/g). The median BPA content in recycled textiles (13.5 ng/g) was almost twice as high as that of 7.66 ng/g in conventional textiles. BPS showed a median of 1.85 ng/g in recycled textiles and 3.42 ng/g in conventional textiles, indicating a shift from BPA to BPS in manufacturing practices. Simulated laundry experiments showed an overall reduction in bisphenols concentrations after washing. The study also assessed potential health implications via dermal exposure to dry and sweat-wet textiles compared to a tolerable daily intake (TDI) of 0.2 ng/kg bw/day for BPA set by the European Food Safety Authority (EFSA). Exposure from dry textiles remained below this threshold, while exposure from wet textiles often exceeded it, indicating an increased risk under conditions that simulate sweating or humidity. By finding the widespread presence of bisphenols in textiles, our study emphasises the importance of being aware of the potential risks associated with recycling materials as well as the benefits.

Point-of-care urine tenofovir test predicts future HIV preexposure prophylaxis discontinuation among young users.

Young men who have sex with men and transgender women (YMSM/TGW) have disproportionately high HIV incidence and lower preexposure prophylaxis (PrEP) adherence. Point-of-care (POC) urine tenofovir (TFV) rapid assay (UTRA) testing permits real-time monitoring for nonadherence within clinical settings. We performed UTRA testing among PrEP users to examine the relationship between low PrEP adherence and future PrEP discontinuation, and the accuracy of POC testing compared to gold-standard liquid chromatography tandem mass spectrometry (LC/MS/MS). YMSM/TGW participants ( n = 100) were recruited during a daily PrEP visit. Logistic regression models analyzed the relationship between the primary predictor of urine POC assay results (cutoff 1,500 ng/ml) and the primary outcome of PrEP discontinuation, defined as no PrEP follow-up or prescription within 120 days. Overall, 19% of participants had low urine TFV and 21% discontinued PrEP, while 11% of participants self-reported low PrEP adherence (<4 pills per week), which was only 43% sensitive/84% specific in predicting low TFV levels and was not associated with PrEP discontinuation. Low urine TFV level predicted PrEP discontinuation [adjusted odds ratio (AOR) 6.1; 95% confidence interval (CI): 1.4-11; P = 0.005] and was 71% sensitive/90% specific for discontinuation after 120 days. Compared to LC/MS/MS, UTRA testing had a 98% positive and 100% negative predictive value. In a sample of YMSM/TGW on daily PrEP, POC UTRA testing predicted PrEP discontinuation more accurately than self-reported adherence, with high predictive values compared to LC/MS/MS. UTRA testing may be a clinical tool for directing preventive interventions towards those likelier to discontinue PrEP despite ongoing HIV vulnerability.

An Integrated Strategy for In-depth Profiling of N-acylethanolamines in Biological Samples by UHPLC-HRMS

BackgroundN-acylethanolamines (NAEs) are a class of naturally occurring bioactive lipids that play crucial roles in various physiological processes, particularly exhibiting neuroprotective and anti-inflammatory properties. However, the comprehensive profiling of endogenous NAEs in complex biological matrices is challenging due to their low abundance, structural similarity and the limited availability of commercial standards. Here, we propose an integrated strategy for comprehensive profiling of NAEs that combines chemical derivatization and a three-dimensional (3D) prediction model based on quantitative structure-retention time relationship (QSRR) using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS). ResultsAfter acetyl chloride (ACC) derivatization, the detection sensitivity of NAEs significantly improved. We developed a QSRR prediction model to construct an in-house database for 141 NAEs, encompassing information on RT, MS1 (m/z), and MS/MS spectra. Propargylamine-labeled fatty acids were synthesized as RT calibrants across various analytical conditions to enhance the robustness of the RT prediction model. NAEs in biological samples were then in-depth profiled using parallel reaction monitoring (PRM) acquisition. This integrated strategy identified and annotated a total of 50 NAEs across serum, hippocampus and cortex tissues from a 5xFAD mouse model of Alzheimer's disease. Notably, the levels of polyunsaturated NAEs, particularly NAE 20:5 and NAE 22:6, were significantly decreased in 5xFAD mice compared to WT mice, as confirmed by accurate quantitation using ACC-d0/d3 derivatization. SignificanceOur integrated strategy exhibits great potential for the in-depth profiling of NAEs in complex biological samples, facilitating the elucidation of NAE functions in diverse physiological and pathological processes.

Identification of Key Proteins Related to Cashmere Fiber Diameter by Integrated Proteomics and Bioinformatic Analyses in the Alpas and Alxa Goat Breeds

Background: Goats (Capra hircus) have always been a source of fiber for human use and hold an important place in international high-end textiles. Fiber diameter is the most concerning economic indicator for producers. Understanding the formation mechanism of fiber diameter and its related key proteins can help optimize and control the production of cashmere. Methods: Cashmere goats (n = 36) of the Alpas (n = 18) and Alxa (n = 18) breeds, with a similar age (2 years old) and live weight (25–26 kg), were selected from the Yiwei White Cashmere Goat Breeding Farm, Erdos, Inner Mongolia. Using phenotypic indicators, we evaluated the diameter of the cashmere fibers in Alxa and Alpas goats. We also used electron microscopy to examine the cashmere fiber’s structure and label-free liquid chromatography–tandem mass spectrometry to determine the protein content of the two cashmere fibers. The proteins affecting fiber diameter were identified and analyzed by Western blot, Co-Immunoprecipitation, and bioinformatics analysis. Results: The average diameter of the Alxa breed was smaller (p &lt; 0.05) than that of the Alpas breed (Alxa’s cashmere vs. Alpas’ cashmere). Proteomics technology enabled the highly confident detection of 171 proteins. A total of 68 differentially expressed proteins were identified in the two types of cashmere; 131 proteins were specifically expressed in Alpas goats, and 40 proteins were specifically expressed in Alxa goats. A key protein group that could cause variations in fiber diameter was found using the protein–protein interaction network. To ascertain the reason for the variation in fiber diameter, a structural study of the major protein groups was carried out. Conclusions: KRT10, KRT14, KRT17, and KRT82 are the main proteins impacting the diameter difference, and they have a substantial effect on the average fiber diameter.

Ingestion of the Non-Nutritive Sweetener Erythritol, but Not Glucose, Enhances Platelet Reactivity and Thrombosis Potential in Healthy Volunteers-Brief Report.

Although artificial and non-nutritive sweeteners are widely used and generally recognized as safe by the US and European Union regulatory agencies, there have been no clinical trials to assess either long-term cardiovascular disease risks or short-term cardiovascular disease-relevant phenotypes. Recent studies report that fasting plasma levels of erythritol, a commonly used sweetener, are clinically associated with heightened incident cardiovascular disease risks and enhance thrombosis potential in vitro and in animal models. Effects of dietary erythritol on thrombosis phenotypes in humans have not been examined. Using a prospective interventional study design, we tested the impact of erythritol or glucose consumption on multiple indices of stimulus-dependent platelet responsiveness in healthy volunteers (n=10 per group). Erythritol plasma levels were quantified with liquid chromatography tandem mass spectrometry. Platelet function at baseline and following erythritol or glucose ingestion was assessed via both aggregometry and analysis of granule markers released. Dietary erythritol (30 g), but not glucose (30 g), lead to a >1000-fold increase in erythritol plasma concentration (6480 [5930-7300] versus 3.75 [3.35-3.87] μmol/L; P<0.0001) and exhibited acute enhancement of stimulus-dependent aggregation responses in all subjects, agonists, and doses examined. Erythritol ingestion also enhanced stimulus-dependent release of the platelet dense granule marker serotonin (P<0.0001 for TRAP6 [thrombin activator peptide 6] and P=0.004 for ADP) and the platelet α-granule marker CXCL4 (C-X-C motif ligand-4; P<0.0001 for TRAP6 and P=0.06 for ADP). In contrast, glucose ingestion triggered no significant increases in stimulus-dependent release of either serotonin or CXCL4. Ingestion of a typical quantity of the non-nutritive sweetener erythritol, but not glucose, enhances platelet reactivity in healthy volunteers, raising concerns that erythritol consumption may enhance thrombosis potential. Combined with recent large-scale clinical observational studies and mechanistic cell-based and animal model studies, the present findings suggest that discussion of whether erythritol should be reevaluated as a food additive with the Generally Recognized as Safe designation is warranted. URL: https://www.clinicaltrials.gov; Unique identifier: NCT04731363.

Identification of chemical components of large-leaf yellow tea by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Large-leaf yellow tea, a slightly fermented yellow tea that is unique to China, has a stronger hypoglycemic effect than other tea varieties, such as green and black tea. Research on large-leaf yellow tea has focused on its hypoglycemic effect owing to the lack of comprehensive techniques to characterize its chemical components; thus, its development and further promotion are limited. Therefore, the development of a reliable analytical method to fully characterize the chemical components of large-leaf yellow tea is urgently required. In this study, a reliable strategy based on the data-acquisition technology of ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q TOF/MS) was established to rapidly screen and analyze the main chemical components of large-leaf yellow tea by combining the information of neutral loss groups and characteristic fragment ions. The chromatographic separation experiments were performed on a Waters ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with gradient elution using 0.1% formic acid aqueous solution and acetonitrile as the mobile phases. The flow rate was 0.2 mL/min, the sample volume was 2 μL, and the column temperature was 35 ℃. The mass spectral information of the components in a large-leaf yellow tea solution was collected using the full-information tandem MS (MSE) technique in positive and negative ion modes. The specific chemical components of large-leaf yellow tea was identified as follows. First, a self-established database of tea chemical components was constructed based on the literature. The mass spectral cleavage pathways of different types of compounds in large-leaf yellow tea were then sorted using reference substances, and the characteristics of the fragment ions and neutral loss groups were summarized. The precise mass-to-charge ratio of the target chemical components were then obtained based on the mass spectral information. Finally, the structures of the compounds in large-leaf yellow tea were confirmed based on their chromatographic retention times, mass spectral cleavage pathways, characteristic fragment ions, and neutral loss groups. A total of 87 chemical components, including 10 catechins, 32 flavonoids, 16 phenolic acids, 12 tannins, 6 theaflavins, and 11 compounds in other classes, were identified in large-leaf yellow tea. Representative compounds of various classes, including gallocatechin gallate, quercetin, vitexin, gallic acid, chlorogenic acid, 1,3,6-tri-O-galloyl-β-D-glucose, and theaflavin, were selected, and their characteristic fragment ions and neutral loss groups were investigated in detail to reveal the cleavage pathways of different types of compounds in large-leaf yellow tea. The UPLC-Q TOF/MS method established in this study can comprehensively identify the main chemical components of large-leaf yellow tea in a simple, highly sensitive, stable, and reliable manner. This study provides a scientific basis and data support for the discovery of functional ingredients and quality evaluation of large-leaf yellow tea.

Clinical, Biochemical, and Molecular Characteristics of Filipino Patients with Tyrosinemia Type 1

Hereditary tyrosinemia type I (HT1), or hepatorenal tyrosinemia, is an amino acid disorder which may cause hepatic failure as well as renal and neurologic comorbidities. Early detection of this disorder is possible with newborn screening (NBS). The objective of this study is to describe the clinical, biochemical, and molecular characteristics of Filipino patients diagnosed with HT1 through the expansion of the Philippine NBS program in 2014. There were a total of 16 patients with confirmed HT1 from then until September 2022. Clinical and biochemical data during confirmation and initial evaluation, as well as molecular data, were obtained from the patients’ medical records. The cohort included children between the ages of 18 and 54 months at the time of data collection. The mean age at treatment initiation was 26.8 days. The mean succinylacetone level from dried blood spot sampling using tandem mass spectrometry (MS) was 11.1 µmol/L. Biochemical confirmatory tests via plasma amino acid analysis showed mean levels of tyrosine, phenylalanine, and methionine of 506.1 µmol/L, 111.5 µmol/L, and 125.4 µmol/L, respectively. Upon urine organic acid (UOA) analysis, succinylacetone was detected in all except for one patient, who was managed prior to UOA analysis. The most common clinical characteristics were abnormal clotting times (62.5%), elevated alpha fetoprotein (37.5%), anemia (31.3%), and metabolic acidosis (31.3%). The most common genotype was homozygous c.122T&gt;C p.Leu41Pro in 64.3% of patients. The allelic frequency of this pathogenic variant is 71.4%. The inclusion of HT1 in the Philippine NBS program allowed early diagnosis and management of HT1 patients.

Quantitative analysis of phenolics in Trifolium pratense L. flowers and evaluation of antioxidant activity by sensory

Trifolium pratense L. flowers (TPF) were collected and dried in shade in this study. After extraction in methanol, a diluted solution was applied to the liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) device to determine the bioactive compounds quantitatively. Isoquercitrin (38.64 mg/g extract), coumarin (13.66 mg/g extract), and catechin (12.52 mg/g extract) were verified as major products. Antioxidant activity of TPF was performed using a potentiometric PVC membrane sensor to evaluate 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic content of TPF. TPF inhibited the DPPH radical as 31.4 ± 0.02% at the 500-ppm concentration. However, the DPPH activity of gallic acid was determined as 81.43 ± 0.07% and 92.44 ± 0.1% at the TPF concentrations of 12.5 ppm and 25 ppm, respectively. In addition, the total phenolic content was calculated to be 82.4 ± 0.15 mg gallic acid eq/g extract. It was observed that TPF has the potential to be an antioxidant and a valuable source of isoquercitrin, coumarin, and catechin.

Simultaneous determination of Obeticholic acid and its two major metabolites in human plasma after administration of Obeticholic acid tablets using ultra-high performance liquid chromatography tandem mass spectrometry

Obeticholic acid (OCA), a semisynthetic bile acid derivative, was approved for its therapeutic use in primary biliary cirrhosis. OCA has a enterohepatic circulation and host-gut microbiota metabolic interaction, which produce various metabolites. Such metabolites, especially structural isomers of OCA, together with the need to achieve idea lower limit of quantitation (LLOQ) with minimum matrix interference, bring about significant difficulties to the bioanalysis of OCA. Herein, by applying a combination of solid-phase extraction (SPE) and ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), we introduced an approach for the bioanalysis of OCA along with its two major metabolites—glyco-OCA (GOA) and tauro-OCA (TOA) in human plasma, the full validation results of which showed excellent performance. The quantitative range is 0.2506 ∼ 100.2 ng/mL for OCA, 0.2500 ∼ 100.0 ng/mL for GOA, as well as 0.1250 ∼ 50.00 ng/mL for TOA, respectively. This method was successfully applied to the pharmacokinetic studies in healthy subjects following administration of OCA tablets.

The blistering warfare agent O-mustard (agent T) generates protein-adducts with human serum albumin useful for biomedical verification of exposure and forms intramolecular cross-links.

The highly blistering sulfur mustard analogue agent T (bis(2-chloroethylthioethyl) ether), also known as O-mustard or oxy-mustard, is a common impurity in military grade sulfur mustard (SM) and a component of mixtures such as "HT" that are still found in old munitions. Together with sesquimustard (Q), it is the most important SM analogue and tightly regulated as a Schedule 1 chemical under the Chemical Weapons Convention. We report the adducts of T with nucleophilic Cys34 and other residues in human serum albumin (HSA) formed in vitro. A micro liquid chromatography electrospray ionization high-resolution tandem-mass spectrometry method (µLC-ESI MS/HRMS) was developed for the detection and identification of biomarker peptides alkylated by a T-derived hydroxyethylthioethyloxyethylthioethyl (HETEOETE)-moiety (as indicated by an asterisk below). Following proteolysis of T-exposed human plasma with pronase, the dipeptide Cys34*Pro and the single amino acid residue His* were produced. The use of proteinaseK yielded Cys34*ProPhe and theuse of pepsin generated ValThrGlu48*Phe, AlaGlu230*ValSerLysLeu, and LeuGlyMet329*Phe. Corresponding peptide-adducts of SM and Q were detected in a common workflow that in principle allowed the estimation of the mustard or mustard composition encountered during exposure. Novel adducts of Q at the Glu230 and Met239 residues were detected and are reported accordingly. Based on molecular dynamics simulations, we identified regular interactions of the Cys34(-HETEOETE)-moiety with several glutamic acid residues in HSA including Glu86, which is not an obvious interaction partner by visual inspection of the HSA crystal structure. The existence of this and other intramolecular cross-links was experimentally proven for the first time.