High-resolution Liquid Chromatography-tandem Mass Spectrometry Research Articles

In Vivo and In Vitro Metabolic Fate and Urinary Detectability of Five Deschloroketamine Derivatives Studied by Means of Hyphenated Mass Spectrometry.

Ketamine derivatives such as deschloroketamine and deschloro-N-ethyl-ketamine show dissociative and psychoactive properties and their abuse as new psychoactive substances (NPSs) has been reported. Though some information is available on the biotransformation of dissociative NPSs, data on deschloro-N-cyclopropyl-ketamine deschloro-N-isopropyl-ketamine and deschloro-N-propyl-ketamine concerning their biotransformation and, thus, urinary detectability are not available. The aims of the presented work were to study the in vivo phase I and II metabolism; in vitro phase I metabolism, using pooled human liver microsomes (pHLMs); and detectability, within a standard urine screening approach (SUSA), of five deschloroketamine derivatives. Metabolism studies were conducted by collecting urine samples from male Wistar rats over a period of 24 h after their administration at 2 mg/kg body weight. The samples were analyzed using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) and gas chromatography-mass spectrometry (GC-MS). The compounds were mainly metabolized by N-dealkylation, hydroxylation, multiple oxidations, and combinations of these metabolic reactions, as well as glucuronidation and N-acetylation. In total, 29 phase I and 10 phase II metabolites were detected. For the LC-HRMS/MS SUSA, compound-specific metabolites were identified, and suitable screening targets could be recommended and confirmed in pHLMs for all derivatives except for deschloro-N-cyclopropyl-ketamine. Using the GC-MS-based SUSA approach, only non-specific acetylated N-dealkylation metabolites could be detected.

Nontargeted Analysis of Coumarins in Source Water and Their Formation of Chlorinated Coumarins as DBPs in Drinking Water.

Coumarin was detected as one of the most abundant compounds by nontargeted analysis of natural product components in actual water samples prior to disinfection. More importantly, prechlorination of humic acid generated 3-hydroxycoumarin and monohydroxy-monomethyl-substituted coumarin with a total yield of ≤10.1%, which suggested the humic substance in raw water is an important source of coumarins. 7-Hydroxycoumarin, 6-hydroxy-4-methylcoumarin, 6,7-dihydroxycoumarin, and 7-methoxy-4-methylcoumarin were identified in raw water by high-performance liquid chromatography-tandem high-resolution mass spectrometry because only some coumarin standards were commercially available. Their chlorination generated monochlorinated and polychlorinated coumarins, and their structures were confirmed by the synthesized standards. These products could form at various dosages of chlorine and pH levels, and some with a concentration of 600 ng/L can be stable in tap water for days. 3,6,8-Trichloro-7-hydroxycoumarin, 3-chloro-7-methoxy-4-methylcoumarin, and 3,6-dichloro-7-methoxy-4-methylcoumarin were first identified in finished water with concentrations of 0.0670, 78.1, and 14.7 ng/L, respectively, but not in source water, suggesting that they are new DBPs formed during disinfection. The cytotoxicity of 3-chloro-7-methoxy-4-methylcoumarin in CHO-K1 cells was comparable to those of 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone in TIC-Tox analyses, suggesting that further investigation of their occurrence and control in drinking water systems is warranted.

Neuroretinal degeneration in a mouse model of systemic chronic immune activation observed by proteomics.

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.

Biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach using LC-HRMS/MS and chemometrics

Since its first employment in World War I, chlorine gas has often been used as chemical warfare agent. Unfortunately, after suspected release, it is difficult to prove the use of chlorine as a chemical weapon and unambiguous verification is still challenging. Furthermore, similar evidence can be found for exposure to chlorine gas and other, less harmful chlorinating agents. Therefore, the current study aims to use untargeted high resolution mass spectrometric analysis of chlorinated biomarkers together with machine learning techniques to be able to differentiate between exposure of plants to various chlorinating agents. Green spire (Euonymus japonicus), stinging nettle (Urtica dioica), and feathergrass (Stipa tenuifolia) were exposed to 1000 and 7500 ppm chlorine gas and household bleach, pool bleach, and concentrated sodium hypochlorite. After sample preparation and digestion, the samples were analyzed by liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). More than 150 chlorinated compounds including plant fatty acids, proteins, and DNA adducts were tentatively identified. Principal component analysis (PCA) and linear discriminant analysis (LDA) showed clear discrimination between chlorine gas and bleach exposure and grouping of the samples according to chlorine concentration and type of bleach. The identity of a set of novel biomarkers was confirmed using commercially available or synthetic reference standards. Chlorodopamine, dichlorodopamine, and trichlorodopamine were identified as specific markers for chlorine gas exposure. Fenclonine (Cl-Phe), 3-chlorotyrosine (Cl-Tyr), 3,5-dichlorotyrosine (di-Cl-Tyr), and 5-chlorocytosine (Cl-Cyt) were more abundantly present in plants after chlorine contact. In contrast, the DNA adduct 2-amino-6-chloropurine (Cl-Ade) was identified in both types of samples at a similar level. None of these chlorinated biomarkers were observed in untreated samples. The DNA adducts Cl-Cyt and Cl-Ade could clearly be identified even three months after the actual exposure. This study demonstrates the feasibility of forensic biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach.

Integrative leaf anatomy structure, physiology, and metabolome analyses revealed the response to drought stress in sainfoin at the seedling stage.

Sainfoin (Onobrychis viciaefolia) is a vital legume forage, and drought is the primary element impeding sainfoin growth. The anatomical structure, physiological indexes, and metabolites of the leaves of sainfoin seedlings with a drought-resistant line of P1 (DRL) and a drought-sensitive material of 2049 (DSM) were analyzed under drought (-1.0 MPa) with polyethylene glycol-6000 (PEG-6000). The leaf anatomy was studied by the paraffin section method. The related physiological indexes were measured by the hydroxylamine oxidation method, titanium sulfate colorimetric method, thiobarbituric acid method, acidic ninhydrin colorimetric method, and Coomassie brilliant blue method. The metabolomics analysis was composed of liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS). The results revealed that the thickness of the epidermis, palisade tissue, and sponge tissue of DRL were significantly greater than those of DSM. The leaves of DRL exhibited lower levels of superoxide anion (O2 •-) production rate, hydrogen peroxide (H2O2) content, and malondialdehyde (MDA) content compared with DSM, while proline (Pro) content and soluble protein (SP) content were significantly higher than those of DSM. A total of 391 differential metabolites were identified in two samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that the primary differential metabolites were concentrated into the tyrosine metabolism; isoquinoline alkaloid biosynthesis; ubiquinone and other terpenoid quinone biosynthesis; neomycin, kanamycin, and gentamicin biosynthesis; and anthocyanin biosynthesis metabolic pathways. Compared with DSM, DRL had more complete anatomical structure, lower active oxygen content, and higher antioxidant level. The results improved our insights into the drought-resistant mechanisms in sainfoin.

Abstract 1839: Proteomic and glycoproteomic differences associated with prostate cancer in urine

Abstract Background and Aims: Prostate cancer is one of the most common cancers affecting men in the United States, with ~288,300 new cases and 34,700 deaths in 2023 alone. Prostate-specific antigen (PSA), the most widely used prostate cancer biomarker, lacks specificity and sensitivity, leading to unnecessary biopsies, overdiagnosis, and overtreatment. There is an urgent unmet clinical need to find new biomarkers that can distinguish patients with benign disease from those with prostate cancer. Urine is an attractive source for these biomarkers as prostate-derived proteins are more highly concentrated in urine than in blood. To better understand both global proteomic and glycoproteomic molecular signatures that could lead to improved diagnostics for prostate cancer, we analyzed urine samples from unaffected men, men with benign prostatic hyperplasia (BPH), and men with prostate cancer. Methods: Urine samples (N = 58 total) were collected from unaffected men (N = 17), men with BPH (N = 24), and men with prostate cancer (N = 17). Samples were digested with trypsin and analyzed with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for shotgun proteomics analysis. Association with cancer was determined through supervised analysis using Pearson correlation. For glycoproteomics analysis, intact glycopeptides were enriched using strong anion exchange-electrostatic repulsion hydrophilic interaction chromatography, fractionated using high pH reversed-phase chromatography, and analyzed with LC-MS/MS. Association with the three conditions was determined through several rounds of supervised analysis using Pearson correlation. Results: In total, 3542 human proteins were identified, and 78 proteins (p < 0.01) were significantly associated with prostate cancer. The glycolysis pathway and lysosome pathway were enriched in samples from men with prostate cancer. In the glycoproteomics analysis, 36 proteins (p < 0.05) had altered glycan diversity in one of the three conditions, and the complement and coagulation cascade pathway and the lysosome pathway were highly enriched. Six proteins (p < 0.05) had significantly increased glycan diversity in the cancerous samples than in the BPH and normal samples. Conclusions: This study identifies a set of interesting proteomic and glycoproteomic differences in urine from men with prostate cancer compared to BPH and non-cancer samples, which warrant further investigation in additional sample cohorts as potential novel biomarkers. Citation Format: Nikhiya Shamsher, Fernando Garcia-Marques, Abel Bermudez, Mark R. Flory, Sharon J. Pitteri. Proteomic and glycoproteomic differences associated with prostate cancer in urine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1839.

In vitro, invivo metabolism and quantification of the novel synthetic opioid N-piperidinyl etonitazene (etonitazepipne).

N-piperidinyl etonitazene (etonitazepipne) is a newly synthesized opioid related to the 2-benzylbenzimidazole analog class. Etonitazepipne has been formally notified and placed under intensive monitoring in Europe in January 2022. Nitazenes have high affinity at µ-opioid receptor (MOR). Etonitazepipne, specifically shows a EC50 of 2.49 nM, suggesting about 50 times higher potency combined with higher efficacy compared to morphine. Antinociceptive potency l ('hot plate test' with rats) was 192-fold greater than that of morphine. Here we report on a post-mortem case involving etonitazepipne and its quantification using a standard addition method (SAM) through liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, characterization and identification of phase I human metabolites using invitro assay based on pooled human liver microsomes (pHLM) was performed along with the analysis of authentic urine samples by means of high-performance liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). The concentration of etonitazepipne in post-mortem blood and urine was 8.3 and 11 ng/mL, respectively. SAM was validated by assessing the following parameters: intraday and interday repeatability, matrix effect and recovery rate in post-mortem blood. A total of 20 and 14 metabolites were identified after pHLM incubation and urine analysis, respectively. Most pronounced invitro and invivo transformations were O-deethylation, hydroxylation, ketone reduction, and combinations thereof. Considering small traces of the parent drug often found in real cases, the identification of metabolic biomarkers is crucial to identify exposure to this drug. O-deethylated, oxidated metabolites, and combination thereof are proposed as urinary biomarkers along with the parent compound.

Occurrence of perfluoroalkyl substances in canned tuna and their impact on food safety

Continued use for industrial purposes of ever-new compounds compulsorily leads to the possibility of food contamination and risk for the consumer. Among the legacy and emerging contaminants, perfluoroalkyl substances (PFASs), used in the mid-20th century and still being implemented, are molecules of particular concern for their toxicity as endocrine disruptors, immunodepressants, possible carcinogens and liver and kidney toxicity. The main sources of PFAS exposure are water and food, particularly fish. In the European Union, European Food Safety Authority (EFSA) aimed the focus on 4 PFASs: perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexane sulfonic acid (PFHxS) and perfluorooctane sulfonic acid (PFOS). In this study, canned tuna was the investigated matrix, as a highly consumed food. The study aimed to detect the presence of 18 PFASs in 75 paired batches of precooked tuna loins and canned tuna through ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry (UHPLC-HRMS). Seven of the investigated PFASs were detected in the analyzed samples. Among them, just two of the four PFASs of which EFSA established a Tolerable Weekly Intake (TWI), i.e. PFOS and PFNA, were found in canned tuna. The risk characterization related to canned tuna resulted in an intake value much lower than the TWI for the average Italian consumer, but is a possible concern for high consumers, considering that canned tuna is not the only source of PFAS intake.

Targeted screening of the synergistic components in Artemisia annua L. leading to enhanced antiplasmodial potency of artemisinin based on a "top down" PD-PK approach

Ethnopharmacological relevanceArtemisinin (ART) showed enhanced antimalarial potency in the herb Artemisia annua L. (A. annua), from which ART is isolated. Increased absorption of ART with inhibited metabolism in the plant matrix is an underlying mechanism. Several synergistic components have been reported based on a "bottom-up" approach, i.e., traditional isolation followed by pharmacokinetic and/or pharmacodynamic evaluation. Aim of the studyIn this study, we employed a "top-down" approach based on in vivo antimalarial and pharmacokinetic studies to identify synergistic components in A. annua. Materials and methodsTwo A. annua extracts in different chemical composition were obtained by extraction using ethyl acetate (EA) and petroleum ether (PE). The synergistic antimalarial activity of ART in two extracts was compared both in vitro (Plasmodium falciparum) and in vivo (murine Plasmodium yoelii). For the PD-PK correlation analysis, the pharmacokinetic profiles of ART and its major metabolite (ART-M) were investigated in healthy rats after a single oral administration of pure ART (20 mg/kg) or equivalent ART in each A. annua extract. A liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS)-based analytical strategy was then applied for efficient component classification and structural characterization of the differential components in the targeted extract with a higher antimalarial potency. Major components isolated from the targeted extract were then evaluated for their synergistic effect in the same proportion. ResultsCompared with pure ART (ED50, 5.6 mg/kg), ART showed enhanced antimalarial potency in two extracts in vivo (ED50 of EA, 2.9 mg/kg; ED50 of PE, 1.6 mg/kg), but not in vitro (IC50, 15.0–20.0 nM). A significant increase (1.7-fold) in ART absorption (AUC0-t) was found in rats after a single oral dose of equivalent ART in PE but not in EA; however, no significant change in the metabolic capability (AUCART-M/AUCART) was found for ART in either extract. The differential component analysis of the two extracts showed a higher composition of sesquiterpene compounds, especially component AB (3.0% in PE vs. 0.9% in EA) and component AA (14.1% in PE vs. 5.1% in EA). Two target sesquiterpenes were isolated and identified as arteannuin B (AB) and artemisinic acid (AA). The synergism between ART and AB/AA in the same proportion with PE extract (20:1.6:7.6, mg/kg) was verified by a pharmacokinetic study in rats. ConclusionsA "top-down" strategy based on PD-PK studies was successfully employed to identify synergistic components for ART in A. annua. Two sesquiterpene compounds (arteannuin B and artemisinic acid) could enhance the antimalarial potency of ART by increasing its absorption.

Novel PFAS-specific monitoring approach for highly impacted surface waters

In regulatory environmental monitoring programs, only a very small fraction of the vast number of per- and polyfluoroalkyl substances (PFAS) are investigated by target analysis. Therefore, non-target analysis (NTA) studies are increasingly conducted to detect unknown or unnoticed PFAS. These studies are often based on a few grab samples. Thus, discontinuously emitted PFAS from industrial batch processes might be easily overlooked. To address this deficiency and obtain in-depth information on the occurrence and temporal trend of PFAS in surface water impacted by treated industrial waste water, a comprehensive target and NTA study was implemented for 29 months.Elevated PFAS concentrations with up to 10.8 μg L−1 were detected in the river water by target analysis. In addition to PFAS target analysis, the water samples were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). Data processing strategies and various filtering steps were applied to prioritize PFAS. Substances were identified by comparing data to available internal and external PFAS suspect lists, a fragment ion and neutral loss list, and spectral libraries. Several compounds were unequivocally identified based on reference standards.Fifty-five PFAS were (tentatively) identified using NTA. Of those, 43 could be assigned to 13 different homologous series. Partly fluorinated short-chain carboxylic acids (H-PFCA) and sulfonic acids (H-PFSA) were predominantly found in addition to perfluoroalkyl carboxylic acids (PFCA) and the alkyl ether carboxylic acid DONA. To the best of our knowledge, 12 PFAS were reported in surface water for the first time. Signal intensities of individual PFAS and signal ratios varied widely over time, which may indicate batch operations leading to discontinuous emission. Results and insights from this screening approach on PFAS can be used to optimize forthcoming surface water monitoring programs by including newly identified PFAS and selecting appropriate sampling intervals.

Untargeted metabolomics to expand the chemical space of the marine diatom Skeletonema marinoi.

Diatoms (Bacillariophyceae) are aquatic photosynthetic microalgae with an ecological role as primary producers in the aquatic food web. They account substantially for global carbon, nitrogen, and silicon cycling. Elucidating the chemical space of diatoms is crucial to understanding their physiology and ecology. To expand the known chemical space of a cosmopolitan marine diatom, Skeletonema marinoi, we performed High-Resolution Liquid Chromatography-Tandem Mass Spectrometry (LC-MS2) for untargeted metabolomics data acquisition. The spectral data from LC-MS2 was used as input for the Metabolome Annotation Workflow (MAW) to obtain putative annotations for all measured features. A suspect list of metabolites previously identified in the Skeletonema spp. was generated to verify the results. These known metabolites were then added to the putative candidate list from LC-MS2 data to represent an expanded catalog of 1970 metabolites estimated to be produced by S. marinoi. The most prevalent chemical superclasses, based on the ChemONT ontology in this expanded dataset, were organic acids and derivatives, organoheterocyclic compounds, lipids and lipid-like molecules, and organic oxygen compounds. The metabolic profile from this study can aid the bioprospecting of marine microalgae for medicine, biofuel production, agriculture, and environmental conservation. The proposed analysis can be applicable for assessing the chemical space of other microalgae, which can also provide molecular insights into the interaction between marine organisms and their role in the functioning of ecosystems.

Proteomics Landscape and Machine Learning Predictor of Response to Splenectomy in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an auto-immune disease characterized by antibody-mediated platelet destruction. Splenectomy remains the effective curative treatment for patients with ITP who failure to respond to TPO-RAs and/or rituximab (Mageau et al, Am J Hematol. 2022). The effectiveness rate of splenectomy for ITP is reported to be roughly 80%, with a 5-year remission rate ranging from 50% to 75% (Chaturvedi et al, Blood. 2018). For subjects presenting with severely low platelet counts or active bleeding, splenectomy represents the primary approach for rapidly increasing platelet levels and potentially saving lives. To achieve personalized and precise treatment, the prediction efficacy on splenectomy remains an urgent problem. Here, we have characterized immune features of ITP patients with splenectomy, and developed a predictive model for assessing the response to splenectomy. Methods Bone marrow plasma samples before splenectomy were collected and analyzed by high-resolution liquid chromatography-tandem mass spectrometry. Weight protein co-expression network analysis (WPCNA) was applied to generate modules from all proteins and mapped them to clinical characteristics. We used least absolute shrinkage and selection operator (Lasso) regression, recursive feature elimination (RFE) for predictors selection. Next, multivariable logistic regression (LR), support vector machine (SVM), and decision tree algorithms were used to develop a predictive model. Results 60 patients with ITP undergoing splenectomy treatment were enrolled into our study, and divided into three groups according to the response [patients with CR (n=30), NR (n=7), and Relapse (n=23)]. A pathway enrichment analysis of WPCNA modules by ClueGO revealed that megakaryocyte differentiation, humoral immune response mediated by circulating immunoglobulin, Fc receptor signaling pathway, and cGMP-PKG signaling pathway were correspondingly enriched in MEtan, MEturquoise, MEblue, and MEpink modules, which were associated with treatments with TPO-RA, IVIG, rhTPO, and post-operation platelet level, respectively. We then performed KEGG pathway enrichment analyses on the different expression proteins (DEPs) to deciphering the immune landscape among each group. For the relapse group, DEPs were primarily enriched in ribosome, spliceosome, neutrophil extracellular trap formation, and necroptosis pathways, comparing with the CR group. For the NR group, most DEPs were enriched in natural killer cell mediated cytotoxicity, NF-kappa B signaling, Fc gamma R-mediated phagocytosis pathways, comparing with the CR group. The models were based on a multi-step predictor pipeline. Inside the pipeline, features were first filtered by lasso regression and RFE algorithm. We identified five proteomic features present in the bone marrow plasm that associated with relapsed to therapy, which included CD40, COL4A3, GALNT6, GSTM3, and UBE2A (Figure). Each ensemble consisted of three algorithms acting in parallel: LR, SVM and decision tree. The AUC values of LR, SVM, and decision tree were 0.962 (95%CI: 0.915-1), 0.957 (95%CI: 0.897-1), and 0.875 (95%CI: 0.782-0.967), respectively. The three algorithms were then tested for performance. The sensitivity, specificity, positive predictive value, and negative predictive value analysis revealed that LR model reached the best performance. Targeting CD40 represents a promising therapeutic approach for enhancing clinical outcomes in patients with ITP who have shown inadequate response following splenectomy. Conclusion Our analysis revealed that ITP patients with splenectomy showed distinct immune characteristics when achieving different response. Protein features extracted from bone marrow plasma can be selected as predictors of response to splenectomy.

Evaluating membrane bioreactor treatment for the elimination of emerging contaminants using different analytical methods

Since wastewater treatment plants (WWTPs) were not originally designed to eliminate contaminants of emerging concern (CECs), alternative strategies like membrane bioreactor (MBR) technology are gaining importance in achieving effective CEC removal and minimising their environmental impact. In this study, composite wastewater samples were collected from the biggest WWTP in the Basque Country (Galindo, Biscay) and the performance of two secondary treatments (i.e. conventional activated sludge treatment, CAS, and MBR) was assessed. The combination of a suspect screening approach using liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS) and multitarget analysis by gas chromatography-mass spectrometry (GC-MS) allowed the detection of approximately 200 compounds in the WWTP effluents. The estimated removal efficiencies (REs) revealed that only 16 micropollutants exhibited enhanced removal by MBR treatment (RE > 70% or 40 – 60%). The environmental risk posed by the non-eliminated compounds after both treatments remained similar, being anthracene, clarithromycin, bis(2-ethylhexyl) phthalate (DEHP) and dilantin the most concerning pollutants (RQ > 1). The Microtox® bioassay confirmed the MBR’s efficiency in removing baseline toxicity, while suggesting a similar performance of CAS treatment. These minimal differences between treatments call into question the worthiness of MBR treatment and emphasise the need to seek more efficient alternative treatment methods.

Comprehensive characterization of theabrownin polymer structure in Tibetan tea

The intricate molecular structure of theabrownin in Tibetan tea has presented challenges in comprehending its chemical composition. To address this, our study conducted a thorough investigation into the structural features of theabrownin using advanced analytical techniques. Gel permeation chromatography confirmed theabrownin's classification as polymers, with an average molecular weight of 6200. Additionally, Py-GC/MS analysis identified 83 volatile compounds resulting from the thermal degradation of theabrownin, including esters, aldehydes, and phenols. Importantly, hydrolysis experiments, combined with high-resolution liquid chromatography-tandem mass spectrometry analysis, emphasized the crucial role of hydrochloric acid and thiolysis hydrolysis techniques in precisely elucidating the complex composition and structure of theabrownin. The hydrolyzed products verified theabrownin's composition, including jaceosidin, triethyl citrate, α-naphthoflavone, epicatechin, voriconazole, quercetin, and apigenin 7-rutinoside. These findings offer valuable insights that establish a strong scientific foundation for enhancing the quality control and refining the production processes of Tibetan tea.

Increased Circulating IL-32 Is Associated With Placenta Macrophage-derived IL-32 and Gestational Diabetes Mellitus.

Placenta-derived inflammation plays a vital role in the pathophysiology of gestational diabetes mellitus (GDM). IL-32 is a novel pro-inflammatory cytokine and metabolic regulator involved in the development of metabolic disease. We investigated the effect of IL-32 in GDM. First-trimester C-reactive protein (CRP) level was monitored in a case-control study of 186 women with GDM and 186 women without. Placental tissue was lysed and analyzed by high-resolution liquid chromatography-tandem mass spectrometry. Circulating level of inflammatory cytokines IL-32, IL-6, and TNF-α were measured by ELISA kits. The expression of placenta-derived macrophages, inflammatory cytokines, and related pathway proteins were assessed by reverse transcriptase-quantitative PCR, western blot, immunohistochemistry, or immunofluorescence. First-trimester CRP level in peripheral blood was closely associated with glucose and insulin resistance index and was an independent correlation with the development of GDM. High-resolution liquid chromatography-tandem mass spectrometry revealed that placenta-derived CRP expression was dramatically elevated in women with GDM. Interestingly, the expression of placenta-derived IL-32 was also increased and located in the macrophages of placental tissue. Meanwhile, the expression of IL-6, TNF-α, and p-p38 were up-regulated in the placental tissues with GDM. Either IL-6 or TNF-α was colocated with IL-32 in the placental tissue. Importantly, circulating IL-32 throughout pregnancy was increased in GDM and was related to placental-derived IL-32 expression, circulating IL-6, and TNF-α, glucose and insulin resistance index. Increased circulating IL-32 throughout pregnancy was closely associated with placenta macrophage-derived IL-32 expression and GDM. First trimester IL-32 level in peripheral blood may serve to predict the development of GDM.

Metabolism Study of Anamorelin, a GHSR1a Receptor Agonist Potentially Misused in Sport, with Human Hepatocytes and LC-HRMS/MS.

Anamorelin, developed for the treatment of cancer cachexia, is an orally active medication that improves appetite and food intake, thereby increasing body mass and physical functioning. It is classified as a growth hormone secretagogue and strictly monitored by the World Anti-Doping Agency (WADA), owing to its anabolic enhancing potential. Identifying anamorelin and/or metabolite biomarkers of consumption is critical in doping controls. However, there are currently no data available on anamorelin human metabolic fate. The aim of this study was to investigate and identify biomarkers characteristic of anamorelin intake using in silico metabolite predictions with GLORYx, in vitro incubation with 10-donor-pooled human hepatocytes, liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis, and data processing with Thermo Scientific's Compound Discoverer. In silico prediction resulted in N-acetylation at the methylalanyl group as the main transformation (score, 88%). Others including hydroxylation at the indole substructure, and oxidation and N-demethylation at the trimethylhydrazino group were predicted (score, ≤36%). Hepatocyte incubations resulted in 14 phase I metabolites formed through N-demethylation at the trimethylhydrazino group, N-dealkylation at the piperidine ring, and oxidation at the indole and methylalanyl groups; and two phase II glucuronide conjugates occurring at the indole. We propose four metabolites detected as specific biomarkers for toxicological screening.

Liquid chromatography–high-resolution tandem mass spectrometry of anatoxins, including new conjugates and reduction products

Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins for which only a handful of structural analogues have been well characterized. Here, we report the development of an LC–HRMS/MS method for the comprehensive detection of ATXs. Application of this method to samples of benthic cyanobacterial mats and laboratory cultures showed detection of several new ATXs. Many of these result from nucleophilic addition to the olefinic bond of the α,β-unsaturated ketone functional group of anatoxin-a (ATX) and homoanatoxin-a (hATX), analogous to the conjugation chemistry of microcystins, which contain similar α,β-unsaturated amide functionality. Conjugates with glutathione, γ-glutamylcysteine, methanethiol, ammonia, methanol and water were detected, as well as putative C-10 alcohol derivatives. Structural confirmation was obtained by simple and selective analytical-scale semisynthetic reactions starting from available ATX standards. Methanol, water and ammonia conjugates were found to result primarily from sample preparation. Reduction products were found to result from enzymatic reactions occurring primarily after cell lysis in laboratory cultures of Kamptonema formosum and Cuspidothrix issatschenkoi. The relative contributions of the identified analogues to the anatoxin profiles in a set of 22 benthic-cyanobacterial-mat field samples were estimated, showing conjugates to account for up to 15% of total ATX peak area and 10-hydroxyanatoxins up to 38%. The developed methodology, new analogues and insight into the chemical and enzymatic reactivity of ATXs will enable a more comprehensive study of the class than possible previously.

Investigation of Cox-2 inhibition of Laportea decumana (Roxb). Wedd extract to support its analgesic potential

Ethnopharmacological relevanceItchy leaves Laportea decumana (Roxb). Wedd is an indigenous plant in Maluku, Indonesia, and is used traditionally to treat complaints such as fatigue and joint and muscle pains. Aim of the studyTo provide scientific proof of the analgesic effect of L. decumana ethanolic extract tested in in vivo assays while investigating its bioactive phytochemicals using liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS) profiling and Cox-2 inhibition assay. Materials and methodsTo investigate the analgesic activity of the ethanolic extract, assays were conducted on male mice Balb/c strain by chemical induction using acetic acid (i.p.) and heat induction (hotplate). Mice were divided into six groups consisting of six mice, i.e., the baseline group; positive control group (paracetamol 80 mg/kg BW); groups treated with extracts in dosages of 100, 200, and 400 mg/kg bodyweight (BW); and negative control group (acetic acid 0.6%, i. p.). The crude extract was partitioned with liquid–liquid fractionation to yield hexane, ethyl acetate, and water fractions. The extract and fraction were assayed for Cox-2 enzyme inhibition, and the chemical profiles were analyzed using untargeted LC-HRMS. ResultsThe analgesic assays revealed the dose-dependent effect of the extracts, of the effect of treatment with 400 mg/kg BW was not significantly different with that of paracetamol (p < 0.05). The ethyl acetate fraction showed IC50 of 19.25 μg/mL on Cox-2 inhibition (IC50 celexocib 18.48 μg/mL). LC-HRMS showed a distinctive profile of the ethyl acetate fraction compared with those in the extract and other fractions. ConclusionsThis study presents scientific evidence of the analgesic activity of the L. decumana ethanol extract given orally to experimental animals. Inflammatory inhibition plays a role in the overall analgesic mechanism by Cox-2 inhibition of the extract and all fractions. This finding is also supported by the phytochemical profiles of the extract and fractions, showing the presence of compounds reported elsewhere as anti-inflammatory activity.

Atlantic Salmon Gill Epithelial Cell Line (ASG-10) as a Suitable Model for Xenobiotic Biotransformation.

Fish are exposed to xenobiotics in the water. Uptake occurs mainly through the gills, which function as an exchange point with the environment. The gills' ability to detoxify harmful compounds by biotransformation is an essential protection mechanism. The enormous numbers of waterborne xenobiotics requiring ecotoxicological assessment makes it necessary to replace in vivo fish studies with predictive in vitro models. Here, we have characterized the metabolic capacity of the ASG-10 gill epithelial cell line from Atlantic salmon. Inducible CYP1A expression was confirmed by enzymatic assays and immunoblotting. The activities of important cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes were established using specific substrates and metabolite analysis by liquid chromatography (LC) triple quadrupole mass spectrometry (TQMS). Metabolism of the fish anesthetic benzocaine (BZ) in ASG-10 confirmed esterase and acetyl transferase activities through the production of N-acetylbenzocaine (AcBZ), p-aminobenzoic acid (PABA) and p-acetaminobenzoic acid (AcPABA). Moreover, we were able to determine hydroxylamine benzocaine (BZOH), benzocaine glucuronide (BZGlcA) and hydroxylamine benzocaine glucuronide (BZ(O)GlcA) by LC high-resolution tandem mass spectrometry (HRMS/MS) fragment pattern analysis for the first time. Comparison to metabolite profiles in hepatic fractions, and in plasma of BZ-euthanized salmon, confirmed the suitability of the ASG-10 cell line for investigating biotransformation in gills.

Serum Metabolomic Signatures of Hirschsprung's Disease Based on GC-MS and LC-MS.

Hirschsprung's disease (HSCR) is a congenital digestive tract malformation characterized by the absence of intramural ganglion cells in the myenteric and submucosal plexuses along variable lengths of the gastrointestinal tract. Although the improvement of surgical methods has allowed great progress in the treatment of HSCR, its incidence and postoperative prognosis are still not ideal. The pathogenesis of HSCR remains unclear to date. In this study, metabolomic profiling of HSCR serum samples was performed by an integrated analysis of gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) as well as multivariate statistical analyses. Based on the random forest algorithm and receiver operator characteristic analysis, 21 biomarkers related to HSCR were optimized. Several amino acid metabolism pathways were identified as important disordered pathways of HSCR, among which tryptophan metabolism was crucial. To our knowledge, this is the first serum metabolomics study focusing on HSCR, and it provides a new perspective for explaining the mechanism of HSCR.