Liquid Chromatography-high Resolution Mass Spectrometry Research Articles

Enhanced Surveillance of >1100 Pesticides and Natural Toxins in Food: Harnessing the Capabilities of LC-HRMS for Reliable Identification and Quantification

The consequences of climate change along with diverse food regulations and agricultural practices worldwide are complexifying the occurrence and management of chemical contaminants in food. In this context, we present an ultra-high-performance liquid chromatography high-resolution mass spectrometry (LC-HRMS) approach for the simultaneous identification and quantitation of over 1100 pesticide residues, mycotoxins, and plant toxins in cereals and fruits and vegetables. Analytical conditions were optimized to maximize the scope of the targeted molecules, the reliability of compound identification, and quantification performance within a single method. The method was further transferred and validated in another laboratory to assess its ruggedness. Validation according to the SANTE 11312/2021v2 guidelines showed that 92% and 98% of the molecules fulfill the quantification criteria at the lowest validated level in the cereals and fruits and vegetables groups, respectively. Analysis of fifteen certified reference materials led to a 96% satisfactory rate of z-scores confirming method’s competitiveness. Furthermore, the occurrence of these contaminants was studied in 205 cereals and grains samples collected worldwide. The low µg/kg quantification limits make this LC-HRMS method a valuable tool to ensure compliance toward regulations and to screen for non-regulated substances for which occurrence data are crucial for an appropriate risk evaluation.

Analysis of antioxidant nutrients, anti-HIV and anticancer metabolic fingerprints of Pelargonium quercifolium (L.f) L'Hér

Pelargonium quercifolium is an aromatic herb with a strong resinous scent. It is used traditionally to treat heart disease, hypertension, stress, rheumatism, combat hidden hunger, and inhibit HIV-1. The present study aimed to evaluate the toxicity, anti-HIV, antioxidant nutrients and anticancer potentials of the ethanol extract of P. quercifolium. In this study, high-resolution ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), high-performance liquid chromatography with diode-array detection (HPLC-D AD), xCELLigence real-time cell analyser single plate (RTCA-SP), MTT assays and luciferase-based assay were employed to elucidate the phytochemicals, cytotoxicity, anti-HIV, antioxidant nutrients and anticancer metabolites in P. quercifolium. Results show that the characterized metabolites in P. quercifolium most of which were flavonoids, phenolic derivatives, alkaloids and coumarins could arrest cancer cell proliferation and inhibit HIV infection given the high level of cytotoxicity of the tested plant extract. It was also observed and reported for the first time, the nutrient profile of P. quercifolium is higher than most vegetables consumed as a staple food. This study underpins the potential of using anticancer and antiviral metabolites from leaf extract of P. quercifolium as a pharmacological option.

LC–HRMS Lipidomic Fingerprints in Serbian Cohort of Schizophrenia Patients

Schizophrenia (SCH) is a major mental illness that causes impaired cognitive function and long-term disability, so the requirements for reliable biomarkers for early diagnosis and therapy of SCH are essential. The objective of this work was an untargeted lipidomic study of serum samples from a Serbian cohort including 30 schizophrenia (SCH) patients and 31 non-psychiatric control (C) individuals by applying liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) and chemometric analyses. Principal component analysis (PCA) of all samples indicated no clear separation between SCH and C groups but indicated clear gender separation in the C group. Multivariate statistical analyses (PCA and orthogonal partial least squares discriminant analysis (OPLS-DA)) of gender-differentiated SCH and C groups established forty-nine differential lipids in the differentiation of male SCH (SCH-M) patients and male controls (C-M), while sixty putative biomarkers were identified in the differentiation of female SCH patients (SCH-F) and female controls (C-F). Lipidomic study of gender-differentiated groups, between SCH-M and C-M and between SCH-F and C-F groups, confirmed that lipids metabolism was altered and the content of the majority of the most affected lipid classes, glycerophospholipids (GP), sphingolipids (SP), glycerolipids (GL) and fatty acids (FA), was decreased compared to controls. From differential lipid metabolites with higher content in both SCH-M and SCH-F patients groups compared to their non-psychiatric controls, there were four common lipid molecules: ceramides Cer 34:2, and Cer 34:1, lysophosphatidylcholine LPC 16:0 and triacylglycerol TG 48:2. Significant alteration of lipids metabolism confirmed the importance of metabolic pathways in the pathogenesis of schizophrenia.

Equine metabolic investigation of the phosphodiesterase-4 inhibitor ibudilast as a potential performance enhancer.

Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti-inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography-high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. A total of 20 in vivo metabolites were identified under experimental conditions, including 12 Phase I and 8 Phase II conjugated metabolites. Phase I metabolites were predominantly formed through hydroxylation (mono-, di-, and tri-hydroxylation). Demethylated metabolites were also identified during this investigation. Additionally, the research detected Phase II metabolites conjugated with glucuronic and sulfonic acids. The data presented here can assist in detecting the PDE4 inhibitor ibudilast and uncover its illicit use in competitive sports.

Untargeted metabolomics profiling of tropical marine microalgae in batch culture system with different mobile phase using Liquid Chromatography-Orbitrap High-resolution Mass Spectrometry combined with chemometrics

Microalgae have recently been identified as a valuable source of natural bioactive compounds, with potential applications suggested in areas such as food, animal feed, energy production (biofuels), fine chemicals, and pharmaceuticals. However, their chemical diversity remains largely unexplored and it is necessary to determine their potency for further application. This study will explore the diversity of chemical compound from six tropical marine microalgae with untargeted comparative metabolomics approach using a Liquid Chromatography-Orbitrap High Resolution Mass Spectrometry with different mobile phases. Six microalgae species were isolated from Jakarta Bay, Indonesia. Species identification was carried out using morphological and molecular identification. The isolates Chlorella vulgaris InaCC M205 (C5), Chlorella sp. 12 (C12), Tetraselmis subcordiformis InaCC M206 (T2), Tetraselmis sp. 5 (T5), Nannochloropsis oceanica InaCC 207 (N4), and Porphyridium purpureum (P) were then cultivated using f/2 media in 1 L glass photobioreactors, irradiated with daylight lamps (4000 lux) under a 12 h/12 h photoperiod at 25 0C for until cultures reached the stationary phase. Biomass harvesting and measurements were carried out at the end of the culture period. The freeze-dry biomass then was analyzed using liquid chromatography-high resolution mass spectrometry. The results showed that biomass production on day 12 was not significantly different. Biomass production ranged from 0.8 to 1 g/L, with the highest production obtained from the C5 strain and the lowest from the C12 strain. Chromatography analysis revealed that untargeted metabolomics profiling of microalgae, using water-acetonitrile as mobile phase, identified potential compound markers such as N-Methyl-2-pyrrolidone, l-(+)-Cysteine, TBHQ and L-Phenylalanine, which can differentiate between species. Compound such as Cyromazine, Arachidic acid and 13(S)-HOTrE were the potential compound markers when using Water-Methanol as mobile phase. Our research indicates that the metabolomic profile of microalgae can be used to differentiate species depending on the choice of mobile phase.

Significant metabolic alterations in mouse dams exposed to an environmental mixture of polychlorinated biphenyls (PCBs) during gestation and lactation: Insights into PCB and metabolite profiles

Polychlorinated biphenyls (PCBs) and their metabolites are linked to developmental neurotoxicity, but their levels in the gestational and lactational environment remain unexplored. This study investigated the effects of dietary exposure to the Fox River Mixture (FRM) on serum levels of PCBs and their metabolites in female C57BL/6 J mice. Mice were exposed to 0.1, 1.0, or 6.0 mg/kg body weight/day of FRM beginning two weeks before mating and throughout gestation and lactation. Serum samples collected from the dams at weaning were analyzed using gas chromatograph-tandem mass spectrometry and nontarget liquid chromatography-high resolution mass spectrometry. Results showed complex and dose-dependent differences in PCB and metabolite profiles. Untargeted metabolomics revealed alterations in metabolites involved in glucuronidation. Network analysis suggested disturbances in heme and amino acid metabolism associated with higher chlorinated PCBs. These findings suggested that PCBs and metabolites present in the gestational and lactation environment of mice may contribute to developmental neurotoxicity in rodents.

Exploring the Metabolism of Flubrotizolam, a Potent Thieno-Triazolo Diazepine, Using Human Hepatocytes and High-Resolution Mass Spectrometry.

The abuse of psychoactive substances presents challenges in clinical and forensic toxicology. The emergence of novel and potent drugs that pose significant health risks, in particular towards frequent abusers and users unaware of the ingredients, further complicates the situation. Designer benzodiazepines have become a fast-growing subgroup of these new psychoactive substances (NPSs), and their overdose may potentially turn fatal, especially when combined with other central nervous system depressants. In 2021, flubrotizolam, a potent thieno-triazolo designer benzodiazepine, emerged on the illicit market, available online as a "research chemical". The identification of markers of consumption for this designer benzodiazepine is essential in analytical toxicology, especially in clinical and forensic cases. We therefore aimed to identify biomarkers of flubrotizolam uptake in ten-donor-pooled human hepatocytes, applying liquid chromatography high-resolution mass spectrometry and software-aided data mining supported by in silico prediction tools. Prediction studies resulted in 10 and 13 first- and second-generation metabolites, respectively, mainly transformed through hydroxylation and sulfation, methylation, and glucuronidation reactions. We identified six metabolites after 3 h human hepatocyte incubation: two hydroxylated metabolites (α- and 6-hydroxy-flubrotizolam), two 6-hydroxy-glucuronides, a reduced-hydroxy-N-glucuronide, and an N-glucuronide. We suggest detecting flubrotizolam and its hydroxylated metabolites as markers of consumption after the glucuronide hydrolysis of biological samples. The results are consistent with the in vivo metabolism of brotizolam, a medically used benzodiazepine and a chloro-phenyl analog of flubrotizolam.

Evaluation of Nontargeted Mass Spectral Data Acquisition Strategies for Water Analysis and Toxicity-Based Feature Prioritization by MS2Tox.

The machine-learning tool MS2Tox can prioritize hazardous nontargeted molecular features in environmental waters, by predicting acute fish lethality of unknown molecules based on their MS2 spectra, prior to structural annotation. It has yet to be investigated how the extent of molecular coverage, MS2 spectra quality, and toxicity prediction confidence depend on sample complexity and MS2 data acquisition strategies. We compared two common nontargeted MS2 acquisition strategies with liquid chromatography high-resolution mass spectrometry for structural annotation accuracy by SIRIUS+CSI:FingerID and MS2Tox toxicity prediction of 191 reference chemicals spiked to LC-MS water, groundwater, surface water, and wastewater. Data-dependent acquisition (DDA) resulted in higher rates (19-62%) of correct structural annotations among reference chemicals in all matrices except wastewaters, compared to data-independent acquisition (DIA, 19-50%). However, DIA resulted in higher MS2 detection rates (59-84% DIA, 37-82% DDA), leading to higher true positive rates for spectral library matching, 40-73% compared to 34-72%. DDA resulted in higher MS2Tox toxicity prediction accuracy than DIA, with root-mean-square errors of 0.62 and 0.71 log-mM, respectively. Given the importance of MS2 spectral quality, we introduce a "CombinedConfidence" score to convey relative confidence in MS2Tox predictions and apply this approach to prioritize potentially ecotoxic nontargeted features in environmental waters.

Application of pattern deconvolution strategies for the estimation of bromochloro alkane concentrations in indoor dust samples

Bromochloro alkanes (BCAs) are a class of flame retardants similar in structure to polychlorinated alkanes (PCAs), which are the major component of short-chain chlorinated paraffins (SCCPs) listed as Persistent Organic Pollutants under the Stockholm Convention. BCAs have recently been detected for the first time in environmental samples. Due to the complete lack of commercially available analytical standards, no method for quantifying BCAs has been reported to date. In this study, 16 custom-synthesised standards with mixed bromine and chlorine halogenation and carbon chain lengths ranging from C10 to C17 were characterized by liquid chromatography and Orbitrap high-resolution mass spectrometry and used to assess the applicability of pattern deconvolution quantification strategies for BCAs in indoor dust. Br1-9 and Cl1-8 BCAs were detected as [M + Cl]– adduct ions among the C10 to C17 standards, as well as numerous PCA homologues. After applying correction factors to account for the presence of PCAs in the standards, triplicate fortification experiments using varied halogenation composition and concentration determined an average measurement accuracy of 81% over the carbon chain lengths studied and coefficient of variance ≤20% between replicates. Overall, approximately 89% of the ΣBCA concentrations quantified in the fortification trials met the European Union Reference Laboratory's accuracy acceptability criteria recommended for PCAs, between 50 and 150%. Application of the BCA pattern deconvolution quantification procedure to seven representative indoor dust samples from the United States of America revealed a low correlation between the homologue distribution in the samples and the prototype standards (R2 ≤ 0.40), which precluded reliable quantification. This study indicates that pattern deconvolution is an appropriate strategy for quantifying BCAs in environmental samples, but that a large set of appropriate mixture standards will be required before more reliable estimates of BCA concentrations can be achieved in indoor dust.

Metabolomic Effects of Liraglutide Therapy on the Plasma Metabolomic Profile of Patients with Obesity.

Liraglutide, a long-acting glucagon-like peptide-1 receptor agonist (GLP1RA), is a well-established anti-diabetic drug, has also been approved for the treatment of obesity at a dose of 3 mg. There are a limited number of studies in the literature that have looked at changes in metabolite levels before and after liraglutide treatment in patients with obesity. To this end, in the present study we aimed to explore the changes in the plasma metabolomic profile, using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in patients with obesity. A single-center prospective study was undertaken to evaluate the effectiveness of 3 mg liraglutide therapy in twenty-three patients (M/F: 8/15) with obesity, mean BMI 40.81 ± 5.04 kg/m2, and mean age of 36 ± 10.9 years, in two groups: at baseline (pre-treatment) and after 12 weeks of treatment (post-treatment). An untargeted metabolomic profiling was conducted in plasma from the pre-treatment and post-treatment groups using LC-HRMS, along with bioinformatics analysis using ingenuity pathway analysis (IPA). The metabolomics analysis revealed a significant (FDR p-value ≤ 0.05, FC 1.5) dysregulation of 161 endogenous metabolites (97 upregulated and 64 downregulated) with distinct separation between the two groups. Among the significantly dysregulated metabolites, the majority of them were identified as belonging to the class of oxidized lipids (oxylipins) that includes arachidonic acid and its derivatives, phosphorglycerophosphates, N-acylated amino acids, steroid hormones, and bile acids. The biomarker analysis conducted using MetaboAnalyst showed PGP (a21:0/PG/F1alpha), an oxidized lipid, as the first metabolite among the list of the top 15 biomarkers, followed by cysteine and estrone. The IPA analysis showed that the dysregulated metabolites impacted the pathway related to cell signaling, free radical scavenging, and molecular transport, and were focused around the dysregulation of NF-κB, ERK, MAPK, PKc, VEGF, insulin, and pro-inflammatory cytokine signaling pathways. The findings suggest that liraglutide treatment reduces inflammation and modulates lipid metabolism and oxidative stress. Our study contributes to a better understanding of the drug's multifaceted impact on overall metabolism in patients with obesity.

Per-and polyfluoroalkyl substances and disrupted sleep: mediating roles of proteins

BackgroundPer-and polyfluoroalkyl substances (PFAS) contamination may disrupt sleep through disrupted metabolic and immune functions. The study aims to investigate the association and potential mechanism between PFAS and sleep. MethodsWe included 136 young adults recruited between 2014-2018 and 76 were re-assessed between 2020-2022. Additional 8 participants only had complete data between 2020-2022. Plasma PFAS (PFOS, PFOA, PFHxS, PFHpS, PFPeS, PFNA, PFDA) were measured at both visits using liquid-chromatography high-resolution mass spectrometry. Plasma proteins were measured by Olink® Explore 384 Cardiometabolic and Inflammation Panel I. Sleep duration was self-reported at both visits along with follow-up sleep disturbance and sleep-related impairment using validated instruments. We utilized multiple linear regression to explore the association between individual PFAS (in tertile) and these sleep outcomes. PFAS associated with sleep outcomes were subjected to computational toxicology analysis using the Comparative Toxicogenomics Database and Toxicology in the 21st Century database to identify potential genetic links between them. Mediation analysis using proteomic data was then performed to confirm the findings from computational toxicology analysis. ResultsAt baseline, one tertile increase in PFDA was associated with 0.39 (95 % CI: 0.05, 0.73) hours of shorter nightly sleep duration, and, at follow-up, PFHxS and PFOA were associated with 0.39 (95 % CI: 0.05, 0.72) and 0.32 (95 % CI: 0.01, 0.63) hours shorter sleep duration, respectively. One tertile increase in PFOS exposure was associated with a 2.99-point increase in sleep disturbance scores (95 % CI: 0.67, 5.31) and a 3.35-point increase in sleep-related impairment scores (95 % CI: 0.51, 6.20). Computational toxicology and mediation analyses identified potential mediating roles for several proteins in the PFAS-sleep associations, including 11-beta-dehydrogenase isozyme 1 (HSD11B1), cathepsin B (CTSB) and several immune system-related proteins. ConclusionFuture large scale epidemiological and mechanistic studies should confirm our findings and test effect measure modification of the associations by age.

Determining the Degree of Sulfo-tag Conjugation to AAV5 Vectors by LC-HRMS and Evaluating the Effects on Antibody Binding Affinity and Bridging Assay Sensitivity.

Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.

Bioactivity of Amphidinol-Containing Extracts of Amphidinium carterae Grown Under Varying Cultivation Conditions.

Microalgae are of great interest due to their ability to produce valuable compounds, such as pigments, omega-3 fatty acids, antioxidants, and antimicrobials. The dinoflagellate genus Amphidinium is particularly notable for its amphidinol-like compounds, which exhibit antibacterial and antifungal properties. This study utilized a two-stage cultivation method to grow Amphidinium carterae CCAP 1102/8 under varying conditions, such as blue LED light, increased salinity, and the addition of sodium carbonate or hydrogen peroxide. After cultivation, the biomass was extracted and fractionated using solid-phase extraction, yielding six fractions per treatment. These fractions were analyzed using Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS/MS) to identify their chemical components. Key amphidinol compounds (AM-B, AM-C, AM-22, and AM-A) were identified, with AM-B being the most abundant in Fraction 4, followed by AM-C. Fraction 5 also contained a significant amount of AM-C along with an unknown compound. Fraction 4 returned the highest antimicrobial activity against the pathogens Staphylococcus aureus, Enterococcus faecalis, and Candida albicans, with Minimal Biocidal Concentrations (MBCs) ranging from 1 to 512µg/mL. Results indicate that the modulation of both amphidinol profile and fraction bioactivity can be induced by adjusting the cultivation parameters used to grow two-stage batch cultures of A. carterae.

Multisample lipidomic profiles of irritable bowel syndrome and irritable bowel syndrome-like symptoms in patients with inflammatory bowel disease: new insight into the recognition of the same symptoms in different diseases.

Overlapping clinical manifestations of irritable bowel syndrome (IBS) and IBS-like symptoms in patients with inflammatory bowel disease (IBD-IBS) present challenges in diagnosis and management. Both conditions are associated with alterations in metabolites, but few studies have described the lipid profiles. Our aim was to pinpoint specific lipids that contribute to the pathogenesis of IBS and IBD-IBS by analyzing multiple biologic samples. Diarrhea-predominant IBS (IBS-D) patients (n = 39), ulcerative colitis in remission with IBS-like symptoms patients (UCR-IBS) (n = 21), and healthy volunteers (n = 35) were recruited. IBS-D patients meet the Rome IV diagnostic criteria, and UCR-IBS patients matched mayo scores ≤ two points and Rome IV diagnostic criteria. Serum, feces, and mucosa were collected for further analysis. Lipid extraction was carried out by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Lipidomics of mucosa and serum samples significantly differed among the three groups. Feces showed the most altered lipid species, and the enrichment analysis of 347 differentially abundant metabolites via KEGG pathway analysis revealed that alpha-linolenic acid metabolism was significantly altered in the two groups (P < 0.01). The ratio of omega-6/omega-3 fatty acid were imbalance in serum samples. This study revealed a comprehensive lipid composition pattern between IBS-D patients and UCR-IBS patients. We found several distinctive lipids involved in alpha-linolenic acid metabolism, reflecting an imbalance in the omega-6/omega-3 fatty acid ratio. Compared to mucosa and serum samples, fecal samples might have more advantages in lipidomics studies due to the convenience of sample collection and effectiveness in reflecting metabolic information.

Combining High-Resolution Mass Spectrometry and Chemiluminescence Analysis to Characterize the Composition and Fate of Total N-Nitrosamines in Wastewater Treatment Plants.

Monitoring the prevalence and persistence of N-nitrosamines and their precursors in wastewater treatment plants (WWTPs) and effluent-receiving aquatic compartments is a priority for utilities practicing wastewater recycling or exploiting wastewater-impacted source waters. In this work, we developed an analytical framework that combines liquid chromatography-high-resolution mass spectrometry (LC-HRMS) with acidic triiodide-chemiluminescence analysis to characterize the composition and fate of total N-nitrosamines (TONO) and their precursors along the treatment trains of eight WWTPs in New York. Through the parallel application of LC-HRMS and chemiluminescence methods, the TONO scores for 41 N-nitrosamines containing structurally diverse substituents on their amine nitrogen were derived based on their solid-phase extraction recoveries and conversion efficiencies to nitric oxide. Correcting the compositional analysis of TONO using the TONO scores of target N-nitrosamines refined the assessment of the reduction or accumulation of TONO and their precursors across treatment steps in WWTPs. Nontargeted analysis prioritized seven additional N-nitrosamines for confirmation by reference standards, including three previously uncharacterized species: N-nitroso-tert-butylphenylamine, N-nitroso-2-pyrrolidinmethanol, and N-nitrosodesloratadine, although they only served as minor components of TONO. Overall, our study establishes an adaptable methodological framework for advancing the quantitative and qualitative analysis of specific and unknown components of TONO across water treatment and reuse scenarios.

Exploring methandienone metabolites generated via homogenized camel liver: Advancements for anti-doping applications through High Resolution-Liquid Chromatography Mass Spectrometry analysis.

Anabolic steroids, also known as anabolic-androgenic steroids (AAS), encompass steroidal androgens such as testosterone, as well as synthetic counterparts with similar structures and effects. The misuse of AAS has increased over the years, leading to ethical and welfare concerns in sports. The World Anti-Doping Agency (WADA) and the International Federation for Equestrian Sports (FEI) have banned AAS in relevant sports. Methandienone is one of the most identified anabolic androgenic steroids in sports drug testing, Therefore, reliable detection methods are crucial for effective doping control and maintaining the integrity of the sports. This study explores the use of homogenized camel liver for detecting methandienone metabolites in camels. The biotransformation pathways of methandienone in homogenized camel liver tissues are analyzed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) to identify and characterize the phase I and phase II metabolites. Chromatographic separation was achieved using a Thermo-Hypersil C18 column. The study has identified 11 methandienone metabolites (M1-M11), this includes 10 phase I and one phase II metabolite. A glucuronic acid conjugate of methandienone was observed in this study, but no sulfonic acid conjugations were found. The metabolites and their possible chemical structures, along with their fragmentation patterns are confirmed using MSMS (MS2) experiments in data-independent acquisition (DIA) mode. These findings serve as a vital tool for the rapid detection of methandienone, combating its illicit use in camel racing. Comprehensive screenings covering both the parent drug and its metabolites are recommended to improve detection accuracy and ensure regulatory compliance in sports doping. Future research should explore methandienone's metabolite profile in administered camel samples.

Liquid chromatography-high-resolution mass spectrometry-based metabolomics revealing the effects of zearalenone and alpha-zearalenol on human endometrial cancer cells.

Human exposure to mycotoxins through food involve a mixture of compounds, which can be harmful to human health. The Fusarium fungal species are known to produce zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, and its metabolite alpha-zearalenol (α-ZEL), both of which possess endocrine-disruptive properties. Given their potential harm to human health through food exposure, investigating the combined effects of ZEN and α-ZEL becomes crucial. Hence, the combined impact of ZEN and α-ZEL study hold significant importance. This in vitro study delves into the critical area, examining their combined impact on the proliferation and metabolic profile of endometrial cancer Ishikawa cells via sulforhodamine, clonogenic, proliferating cell nuclear antigen (PCNA) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) based untargeted metabolomics. Low concentrations of ZEN (25nm), α-ZEL (10nm), or a combination of both were observed to significantly enhance cell proliferation of Ishikawa cells, as evidenced by PCNA immunostaining, immunoblotting as well and clonogenic assays. The metabolomics revealed the perturbations in glycerophospholipid metabolism, nicotinate and nicotinamide metabolism and phenylalanine, tyrosine, tryptophan biosynthesis provides valuable insights into potential mechanism by which these mycotoxins may facilitate cell proliferation. However, further investigations are warranted to comprehensively understand the implications of these findings and their possible implications for human health.

Profiling organic pollutants in environmental water by dansylation-based non-targeted liquid chromatography-high resolution mass spectrometry analysis

Monitoring organic pollutants in rainwater is important to understand relations between air pollutants and water safety. Non-targeted liquid chromatography–mass spectrometry (LC–MS) is a powerful tool but because of big differences of pollutants in polarity, ionization efficiency, and low concentrations, it is challenging to detect all pollutants in a single analysis. Chemical derivatization is a widely adapted strategy to fractionate complex samples with enhanced sensitivity and selectivity. Herein, we propose the usage of dansylation as a chemical derivatization method to improve both LC retention and MS ionization of organic pollutants containing amine, hydroxyl, and carboxyl for non-targeted LC–MS analysis. We first evaluated the labeling coverage of dansylation to organic pollutants in water matrix. Using dansyl chloride (DnsCl) and dansyl hydrazine (DnsHz) to label 100 amine- and hydroxyl-containing compounds and 100 carboxyl compounds, respectively, we found DnsCl and DnsHz had over 60% labeling coverage for 8 categories of compounds. Then dansylation was applied to label the rainwater, source water, disinfected rainwater, and drinking water samples. To facilitate the annotation of dansylated compounds, we also established a web-based tool termed DansylFinder. Using DansylFinder, 3889, 5813, 6077, and 4050 tentative annotations were found in rainwater, source water, disinfected rainwater, and drinking water samples by dansylation-based non-targeted LC-HRMS analysis. Four hundred fifty four were persistently detected in all water samples, suggesting significant organic overlaps of the four water samples. In addition, two degradation pathways reported in drinking water disinfection process were also detected in disinfected rainwater, suggesting rainwater is a potential path for air pollutants to infiltrate drinking water system.

Differences in Prediagnostic Serum Metabolomic and Lipidomic Profiles Between Cirrhosis Patients with and without Incident Hepatocellular Carcinoma.

Early detection of hepatocellular carcinoma (HCC) is crucial for improving patient outcomes, but we lack robust clinical biomarkers. This study aimed to identify a metabolite and/or lipid panel for early HCC detection. We developed a high-resolution liquid chromatography mass spectrometry (LC-MS)-based profiling platform and evaluated differences in the global metabolome and lipidome between 28 pre-diagnostic serum samples from patients with cirrhosis who subsequently developed HCC (cases) and 30 samples from patients with cirrhosis and no HCC (controls). We linked differentially expressed metabolites and lipids to their associated genes, proteins, and transcriptomic signatures in publicly available datasets. We used machine learning models to identify a minimal panel to distinguish between cases and controls. Among cases compared with controls, 124 metabolites and 246 lipids were upregulated, while 208 metabolites and 73 lipids were downregulated. The top upregulated metabolites were glycoursodeoxycholic acid, 5-methyltetrahydrofolic acid, octanoyl-coenzyme A, and glycocholic acid. Elevated lipids comprised glycerol lipids, cardiolipin, and phosphatidylethanolamine, whereas suppressed lipids included oxidized phosphatidylcholine and lysophospholipids. There was an overlap between differentially expressed metabolites and lipids and previously published transcriptomic signatures, illustrating an association with liver disease severity. A panel of 12 metabolites that distinguished between cases and controls with an area under the receiver operating curve of 0.98 for the support vector machine (interquartile range, 0.9-1). Using prediagnostic serum samples, we identified a promising metabolites panel that accurately identifies patients with cirrhosis who progressed to HCC. Further validation of this panel is required.

Unraveling the fate of 6PPD-Q in aquatic environment: Insights into formation, dissipation, and transformation under natural conditions

The widespread occurrence of N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-Q) in aquatic environments and its hazards to aquatic species underscore the necessity of comprehending its environmental fate. Here, we investigated the transformation from 6PPD to 6PPD-Q and the attenuation of 6PPD-Q in surface water under natural conditions. Contrary to prior findings, this work revealed that 6PPD-Q and its precursor 6PPD-OH/6PPD-(OH)2, were not detected through target analysis and suspect screening during 6PPD transformation in the surface water under the natural conditions. 6PPD-Q predominantly accumulated in TWPs in ambient atmosphere with 1.28 % mass yield from the 6PPD dissipation. Subsequently, 6PPD-Q was eluted from TWPs and released to the water environment. The investigation on the natural attenuation of 6PPD-Q in the surface water demonstrated that direct and indirect photolysis facilitated the rapid dissipation of 6PPD-Q with a half-life of 2.57 h. Utilizing the liquid chromatography high resolution mass spectrometry (LC-HRMS), including both time of flight (TOF) MS and Orbitrap MS, twelve novel transformation products (TPs) of 6PPD-Q were identified by using a comprehensive non-targeted screening strategy. The results from two dimensions gas chromatography (GC×GC) TOF-MS revealed additional two TPs. Based on the molecular structure of TPs, four major pathways of 6PPD-Q attenuation were proposed, including bond cleavage, hydroxylation, quinone cleavage and rearrangement. All TPs were predicted to exhibit lower toxicity, indicating the natural attenuation of 6PPD-Q reduced its toxicity and potential environmental risks. This study provides crucial insights into the environmental fate of 6PPD-Q, highlighting the significance of understanding both its formation from 6PPD and its subsequent attenuation processes under natural conditions.