Liquid Chromatography-high Resolution Mass Spectrometry Research Articles

Plasma lipidome differences in patients with and without significant carotid plaque

BackgroundAtherosclerosis is a major cause of ischemic stroke, and early detection of advanced atherosclerosis in the carotid artery is important for reducing morbidity and mortality. What is even more important is not only detection of atherosclerosis but early determination whether the patients are at high risk of an event with adverse effects as the size of the plaque does not necessarily reflect its potential to trigger such events. AimWe studied whether plasma lipidomics profile can be used as a diagnostic tool for stratification of stable or unstable plaques without the need of removing the carotid plaque. MethodsThis study used liquid chromatography high-resolution tandem mass spectrometry lipidomics to characterize lipid profiles in patients' plasma and found that patients with significant and complicated (vulnerable) atherosclerotic plaque had distinct lipid profiles compared to those with insignificant plaques. ResultsThe lipid classes that were most predictive of vulnerable plaque were lysophosphoethanolamines, fatty acyl esters of hydroxy fatty acids, free fatty acids, plasmalogens, and triacylglycerols. Most of these compounds were found decreased in plasma of patients with unstable plaques which enabled sufficient performance of a statistical model used for patient stratification. ConclusionsPlasma lipidomes measured by liquid chromatography-mass spectrometry show differences in patients with stable and unstable carotid plaques, therefore these compounds could potentially be used as biomarkers for unstable plaque in future clinical diagnosis.

Abstract PO4-07-02: Development of an Artificial Intelligence-based breast cancer detection model using Plasma Lipidomic Signature

Abstract Background: Mammography is the current diagnostic standard for breast cancer screening and monitoring. However, accessibility challenges, accuracy issues and patient discomfort all contribute to reduced patient compliance and utilization, resulting in a need for more effective diagnostic tools. An artificial intelligence (AI)-based lipidomic blood test may add significant value to early breast cancer detection rate and improve outcomes for patients. We have previously reported a series of lipidomic studies (n=793) and derived a lipid signature from plasma-enriched extracellular vesicles (EVs) that effectively distinguished people with localized breast cancer from cancer-free controls. Here we report the development of a breast cancer detection AI model from lipidomic data assessed directly using plasma samples. Methods: Lipids in both EVs and plasma collected from fasted breast cancer and control blood samples (n=256) were extracted and analysed by liquid chromatography-high resolution mass spectrometry (LC-HRAM-MS). Over 400 manually curated lipids were quantified. A bootstrapped analysis using Boruta, a robust and statistically rigorous feature selection algorithm based on random forest feature importance, was employed to identify cancer discriminatory lipid signatures in EV and plasma lipidomes consistently selected across 2000 bootstrap samples. The resulting lipid signature was then used to train an ensemble of 18 distinct machine learning models for cancer status prediction using a majority vote to aggregate the individual predictions. Model performance and variability were assessed over 2000 iterations of leave-group-out cross-validation (LGOCV) using an 80/20 train-test split. Average patient-level predictions across LGOCV iterations were recorded for both EV-and plasma-derived models and the two modalities were compared using an exact paired samples test (McNemar’s test). Results: Both the EV- and plasma-derived lipid signatures performed well in distinguishing breast cancer samples from controls. The development of a bioinformatics AI pipeline enabled the creation of a robust ensemble model achieving an F1 score of 0.89 in plasma with LGOCV. The final plasma ensemble predictive performance of 86.1% (±4.5%) in accuracy, 91.4% (±5.4%) in sensitivity, and 78.7% (±8.6%) specificity was achieved, which is comparable to that of EV (accuracy: 86.1±4.4%, sensitivity: 90.4±5.3%, specificity: 80.2±8.7%). Paired samples analysis using McNemar’s test indicated no significant differences between models trained on EV- and plasma-derived lipid signatures in either the sensitivity (p=0.65), specificity (p=0.49), or accuracy (p=0.42). Conclusion: The initial study demonstrated the high performance of a plasma-enriched extracellular vesicle-derived lipid biomarker signature for early breast cancer detection. Direct assessment of the lipidomic signature from plasma showed promise in simplifying the test. Assessing plasma directly offered advantages in terms of scalability, higher throughput, and ease of implementation. Further verification of the lipid signature in an upcoming study involving 500 plasma samples is planned. Ongoing studies will further optimize the plasma lipidomic signature and strengthen our AI pipeline. These findings support the potential clinical application of AI-based lipidomic profiling as a blood-based screening tool for breast cancer detection. Citation Format: Ameline Lim, Cheka Kehelpannala, Fatemeh Vafaee, Forrest Koch, Dana Pascovici, Desmond Li, Kerry Heffernan, Gillian Lamoury, Amani Batarseh, Bruce Mann. Development of an Artificial Intelligence-based breast cancer detection model using Plasma Lipidomic Signature [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-07-02.

Untargeted metabolomics using liquid chromatography-high resolution mass spectrometry and chemometrics for analysis of non-halal meats adulteration in beef meat.

The adulteration of raw beef (BMr) with dog meat (DMr) and pork (PMr) becomes a serious problem because it is associated with halal status, quality, and safety of meats. This research aimed to develop an effective authentication method to detect non-halal meats (dog meat and pork) in beef using metabolomics approach. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) using untargeted approach combined with chemometrics was applied for analysis non-halal meats in BMr. The untargeted metabolomics approach successfully identified various metabolites in BMr DMr, PMr, and their mixtures. The discrimination and classification between authentic BMr and those adulterated with DMr and PMr were successfully determined using partial least square-discriminant analysis (PLS-DA) with high accuracy. All BMr samples containing non-halal meats could be differentiated from authentic BMr. A number of discriminating metabolites with potential as biomarkers to discriminate BMr in the mixtures with DMr and PMr could be identified from the analysis of variable importance for projection value. Partial least square (PLS) and orthogonal PLS (OPLS) regression using discriminating metabolites showed high accuracy (R2>0.990) and high precision (both RMSEC and RMSEE <5%) in predicting the concentration of DMr and PMr present in beef indicating that the discriminating metabolites were good predictors. The developed untargeted LC-HRMS metabolomics and chemometrics successfully identified non-halal meats adulteration (DMr and PMr) in beef with high sensitivity up to 0.1% (w/w). A combination of LC-HRMS untargeted metabolomic and chemometrics promises to be an effective analytical technique for halal authenticity testing of meats. This method could be further standardized and proposed as a method for halal authentication of meats.

The screening method for 39 phytotoxins and mycotoxins in blood and urine with liquid chromatography-high resolution mass spectrometry

BackgroundPoisonings caused by plant toxins and mycotoxins occur frequently, which do great harm to human health and social public health safety. When a poisoning incident occurs, biological samples are commonly be used to conduct the detection of toxic substances and their metabolites for targeted clinical treatment and incident analysis. ObjectiveTo establish an efficient and accurate analysis method of 39 phytotoxins and mycotoxins in blood and urine by high performance liquid chromatography quadrupole tandem orbitrap mass spectrometry (HPLC-Orbitrap MS). MethodAfter 3 mL of methanol being added to 1 mL blood and urine respectively for extraction and protein precipitation, the supernatant was injected into HPLC-Orbitrap MS for analysis. The phytotoxins and mycotoxins were separated by Hypersil GOLD PFP column with gradient elution using methanol-5 mmol/L ammonium acetate as mobile phase. The data were collected in ESI positive ion mode using Full MS/dd-MS2 for mass spectrometry detection. ResultThe mass database of 39 phytotoxins and mycotoxins was developed, and accurate qualitative analysis can be obtained by matching with the database using the proposed identification criteria. Limit of detections (LODs) were 1.34 × 10−4 ∼ 1.92 ng/mL and 1.92 × 10−4 ∼ 9.80 ng/mL for blood and urine samples, respectively. Limits of quantification (LOQ) of toxins in blood and urine ranged from 4.47 × 10−4 ∼ 6.32 ng/mL and 6.39 × 10−4 ∼ 32.67 ng/mL, respectively. Intra-day relative standard deviations (RSDs) were 0.79 % ∼ 10.90 %, and inter-day RSDs were 1.08 % ∼ 18.93 %. The recoveries can reach 90 % ∼ 110 % with matrix matching calibration curves. ConclusionThe established method is simple and rapid to operate, which can complete the sample analysis within 30 min, providing technical support for clinical poisoning treatment and public health poisoning analysis.

Comparison of Three Widely Employed Extraction Methods for Metabolomic Analysis of Trypanosoma brucei.

Trypanosoma brucei (Tb) is the causative agent of human African trypanosomiasis (HAT), also known as sleeping sickness, which can be fatal if left untreated. An understanding of the parasite's cellular metabolism is vital for the discovery of new antitrypanosomal drugs and for disease eradication. Metabolomics can be used to analyze numerous metabolic pathways described as essential to Tb. brucei but has some limitations linked to the metabolites' physicochemical properties and the extraction process. To develop an optimized method for extracting and analyzing Tb. brucei metabolites, we tested the three most commonly used extraction methods, analyzed the extracts by hydrophilic interaction liquid chromatography high-resolution mass spectrometry (HILIC LC-HRMS), and further evaluated the results using quantitative criteria including the number, intensity, reproducibility, and variability of features, as well as qualitative criteria such as the specific coverage of relevant metabolites. Here, we present the resulting protocols for untargeted metabolomic analysis of Tb. brucei using (HILIC LC-HRMS). © 2024 Wiley Periodicals LLC. Basic Protocol 1: Culture of Trypanosoma brucei brucei parasites Basic Protocol 2: Preparation of samples for metabolomic analysis of Trypanosoma brucei brucei Basic Protocol 3: LC-HRMS-based metabolomic data analysis of Trypanosoma brucei brucei.

Fagopyrin F fraction from Fagopyrum tataricum demonstrates photodynamic inactivation of skin infecting bacterium and squamous cell carcinoma (A431) cells.

Photodynamic therapy (PDT) stands out as a noteworthy development as an alternative targeted treatment against skin ailments. While PDT has advanced significantly, research into photo-activatable "Green drugs" derived from plants which are less toxic than the synthetic drugs has not kept pace. This study investigates the potential of Fagopyrin F Containing Fraction (FCF) derived from Fagopyrum tataricum in mediating PDT against Staphylococcus aureus and skin cancer cells (A431). FCF was isolated from the plant extract using thin-layer chromatography, followed by identification of the compound through high-performance liquid chromatography and high-resolution liquid chromatography-mass spectrometry. FCF was tested to determine its antibacterial and anticancer efficacy. Results revealed that FCF-mediated PDT exhibited potent action against S. aureus, significantly reducing bacterial viability (MIC 19.5 μg/100 μL). Moreover, FCF-mediated PDT showed good efficacy against A431 cells, resulting in a notable reduction in cell viability (IC50 29.08μg/mL). Given the known association between S. aureus and squamous cell carcinoma (SCC), FCF shows the potential to effectively target and eradicate both SCC and the related S. aureus present within the lesions. In silico study reveals that Fagopyrin F effectively binds with the epidermal growth factor (EGFR), one among the highly expressed proteins in the A431 cells, with a binding energy of -9.6kcal/mol. The affinity of Fagopyrin F for EGFR on A431 cancer cells along with its cytotoxicity against skin cancer cells while safeguarding the normal cells (L929) plays a major part in the way it targets cancer cells. However, its safety, efficacy, and long-term advantages in treating skin conditions require more investigation, including in vivo investigations and clinical trials.

Dynamic protein composition of Saccharomyces cerevisiae ribosomes in response to multiple stress conditions reflects alterations in translation activity

Ribosomes, intercellular macromolecules responsible for translation in the cell, are composed of RNAs and proteins. While rRNA makes the scaffold of the ribosome and directs the catalytic steps of protein synthesis, ribosomal proteins play a role in the assembly of the subunits and are essential for the proper structure and function of the ribosome. To date researchers identified heterogeneous ribosomes in different developmental and growth stages. We hypothesized that under stress conditions the heterogeneity of the ribosomes may provide means to prepare the cells for quick recovery. Therefore the aim of the study was the identification of heterogeneity of ribosomal proteins within the ribosomes in response to eleven stress conditions in Saccharomyces cerevisiae, by means of a liquid chromatography/high resolution mass spectrometry (LC-HRMS) and translation activity tests. Out of the total of 74 distinct ribosomal proteins identified in the study 14 small ribosomal subunit (RPS) and 8 large ribosomal subunit (RPL) proteins displayed statistically significant differential abundances within the ribosomes under stress. Additionally, significant alterations in the ratios of 7 ribosomal paralog proteins were observed. Accordingly, the translational activity of yeast ribosomes was altered after UV exposure, during sugar starvation, cold shock, high salt, anaerobic conditions, and amino acid starvation.

The effect of extraction method on biological activity and phytochemical content of Artocarpus heterophyllus (jackfruit) leaves extract concurrent with its principal component analysis

Artocapus heterophyllus (jackfruit) is available in abundance in tropical regions. The present study explored the effect of different extraction method i.e. maceration, ultrasonication and both of ultrasonication and maceration on its biological activity (antioxidant, antidiabetes and antibacterial activities) and phytochemical content of Artocarpus heterophyllus (jackfruit) leaves extract. Jackfruit leaves were extracted using ethanol as solvent. The capability of jackfruit leaves extract to scavenge radicals was evaluated using a DPPH (2,2-diphenyl-1-picryl-hydrazyl) assay, β-carotene bleaching assay and reducing power assay. The total phenolic content (TPC) was assessed using the Folin- Ciocalteau method. Its antibacterial activity was assessed using the disc diffusion and microdilution method against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhimurim and Escherichia coli. The α-glucosidase inhibitor assay was conducted using α-glucosidase from Saccharomyces cerevisiae. The results revealed that ulrasonication method give the highest antioxidant activity in three metode used, i.e. DPPH assay with IC50 of 42.51 µg/mL, β-carotene bleaching assay of 57.64%, reducing power assay of 919 mg GAE/g. Meanwhile, for α-glucosidase inhibitory assay, ultrasonication method also had the highest value of 83.44% at concentration of 1000 µg/mL. Furthermore, antibacterial assay using disk diffusion method showed ultrasonication had the highest inhibition zone of 7.81 mm at 5 mg/mL against P. aeruginosa, while maceration method had the highest inhibition zone of 8.02 mm at 5 mg/mL against E. coli. These results in accordance with total phenolic content (TPC) of ultrasonication extract which was the highest of 125.06 mg GAE/g. The liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis suggested that several bioactive compounds such as phenolic, flavonoid and its derivatives was present in the jackfruit leaves extract. Further analysis using principal component analysis (PCA) revealed that the extraction process affected the metabolite composition of the samples which was confirmed by significant separation of the score plots of extraction methods. The present study revealed that jackfruit leaves is a potent source of natural antibacterial, antioxidants and antidiabetic medicines in functional foods and pharmaceutical industries.

The Metabolic and Lipidomic Fingerprint of Torin1 Exposure in Mouse Embryonic Fibroblasts Using Untargeted Metabolomics.

Torin1, a selective kinase inhibitor targeting the mammalian target of rapamycin (mTOR), remains widely used in autophagy research due to its potent autophagy-inducing abilities, regardless of its unspecific properties. Recognizing the impact of mTOR inhibition on metabolism, our objective was to develop a reliable and thorough untargeted metabolomics workflow to study torin1-induced metabolic changes in mouse embryonic fibroblast (MEF) cells. Crucially, our quality assurance and quality control (QA/QC) protocols were designed to increase confidence in the reported findings by reducing the likelihood of false positives, including a validation experiment replicating all experimental steps from sample preparation to data analysis. This study investigated the metabolic fingerprint of torin1 exposure by using liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics platforms. Our workflow identified 67 altered metabolites after torin1 exposure, combining univariate and multivariate statistics and the implementation of a validation experiment. In particular, intracellular ceramides, diglycerides, phosphatidylcholines, phosphatidylethanolamines, glutathione, and 5'-methylthioadenosine were downregulated. Lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, glycerophosphocholine, triglycerides, inosine, and hypoxanthine were upregulated. Further biochemical pathway analyses provided deeper insights into the reported changes. Ultimately, our study provides a valuable workflow that can be implemented for future investigations into the effects of other compounds, including more specific autophagy modulators.

Impact of four different extraction methods and three different reconstitution solvents on the untargeted metabolomics analysis of human and rat urine samples

Unsuitable sample preparation may result in loss of important analytes and consequently affect the outcome of untargeted metabolomics. Due to species differences, different sample preparations may be required within the same biological matrix.The study aimed to compare the in-house sample preparation method for urine with methods from literature and to investigate the transferability of sample preparation from human urine to rat urine. A total of 12 different conditions for protein precipitation were tested, combining four different extraction solvents and three different reconstitution solvents using an untargeted liquid-chromatography high resolution mass spectrometry (LC-HRMS) metabolomics analysis. Evaluation was done based on the impact on feature count, their detectability, as well as the reproducibility of selected compounds.Results showed that a combination of methanol as extraction and acetonitrile/water (75/25) as reconstitution solvent provided improved results at least regarding the total feature count. Additionally, it was found that a higher amount of methanol was most suitable for extraction of rat urine among the tested conditions. In comparison, human urine requires significantly less volume of extraction solvent.Overall, it is recommended to systematically optimize both, the extraction method, and the reconstitution solvent for the used biofluid and the individual analytical settings.

Improved analysis of grape seed extract by liquid chromatography–high resolution mass spectrometry (LC-HRMS) reveals that proanthocyanidin-protein interaction mechanisms in cream depend on degree of polymerization

This study aimed to comprehensively characterize chemical profiles of proanthocyanidins (PACs) from grape seed extract (GSE), examine their interactions with proteins in a cream system, and define the mechanisms mediating PAC-protein interactions. GSE PACs were fractionated and characterized by thiolysis followed by liquid chromatography–high resolution mass spectrometry (LC-HRMS) analysis. New PACs with a degree of polymerization (DP) up to 16 were identified by improved HRMS data processing methods. In the model cream system, high-DP PACs exhibited greater precipitation capacity and protein binding than low-DP PACs. Low-DP PACs primarily engaged in hydrogen bonding, while high-DP PACs predominantly utilized multiple hydrophobic interaction sites to form cream protein aggregates. Furthermore, particle size and viscosity measurement of cream revealed a progressively DP-dependent increase in aggregated fat globules and cream viscosity. These findings enhanced our understanding of PACs’ structural intricacies and highlighted their functional role as PAC-rich natural ingredients in creating structured cream systems.

Deciphering the chemical constituents of Phlomis monocephala extracts using UHPLC-HRMS and their antioxidant, neuroprotective, antidiabetic and toxic potentials

In the current study, five different extracts (n-hexane, ethyl acetate, dichloromethane, ethanol, and water) of Phlomis monocephala were analyzed for the first time by ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) to identify their phenolic compounds. The extracts were also evaluated for their non-enzyme antioxidant activities using a variety of methods, including DPPH and, ABTS•+ scavenging activities, reduction of ferric (Fe3+), and cupric ions (Cu2+), metal chelating (MCA) activities, and phosphomolybdenum (PBD). Additionally, the extracts were assessed for their in vitro enzyme inhibition potential (acetylcholinesterase (AChE)/butyrylcholinesterase (BChE), α-amylase, α-glucosidase, and tyrosinase). Furthermore, cell viability was evaluated on HepG2 (human hepatocellular carcinoma), S17 cells (murine bone marrow stromal) and RAW (murine macrophages). UHPLC-HRMS allowed for the identification of 115 compounds from different chemical groups including flavonoids, iridoid glycosides, phenylethanoid glycosides and others. Most of the extracts had strong antioxidant potential and were rich in phenolic compounds. Ethanol and water extracts appear as the most promising antioxidant extracts providing the highest values followed by ethyl acetate by all the methodologies employed in this study. Furthermore, all the extracts, except the aqueous extract, inhibited all the enzymes significantly. Surprisingly, the aqueous extract did not show a prominent inhibition (low or no inhibition) against these enzymes. Hexane, ethyl acetate, and dichloromethane exhibited extremely significant cytotoxic effects against all cell lines at a higher concentration. The HepG2 cells demonstrated lower sensitivity to the extracts than RAW and S17 cells. In conclusion, P. monocephala can be considered as a valuable ingredient for the production of functional applications including nutraceuticals.

A nested case-control study of untargeted plasma metabolomics and lung cancer among never-smoking women within the prospective Shanghai Women's Health Study.

The etiology of lung cancer in never-smokers remains elusive, despite 15% of lung cancer cases in men and 53% in women worldwide being unrelated to smoking. Here, we aimed to enhance our understanding of lung cancer pathogenesis among never-smokers using untargeted metabolomics. This nested case-control study included 395 never-smoking women who developed lung cancer and 395 matched never-smoking cancer-free women from the prospective Shanghai Women's Health Study with 15,353 metabolic features quantified in pre-diagnostic plasma using liquid chromatography high-resolution mass spectrometry. Recognizing that metabolites often correlate and seldom act independently in biological processes, we utilized a weighted correlation network analysis to agnostically construct 28 network modules of correlated metabolites. Using conditional logistic regression models, we assessed the associations for both metabolic network modules and individual metabolic features with lung cancer, accounting for multiple testing using a false discovery rate (FDR) < 0.20. We identified a network module of 121 features inversely associated with all lung cancer (p = .001, FDR = 0.028) and lung adenocarcinoma (p = .002, FDR = 0.056), where lyso-glycerophospholipids played a key role driving these associations. Another module of 440 features was inversely associated with lung adenocarcinoma (p = .014, FDR = 0.196). Individual metabolites within these network modules were enriched in biological pathways linked to oxidative stress, and energy metabolism. These pathways have been implicated in previous metabolomics studies involving populations exposed to known lung cancer risk factors such as traffic-related air pollution and polycyclic aromatic hydrocarbons. Our results suggest that untargeted plasma metabolomics could provide novel insights into the etiology and risk factors of lung cancer among never-smokers.

Use of carbonyldiimidazole as a derivatization agent for the detection of pinacolyl alcohol, a forensic marker for Soman, by EI-GC-MS and LC-HRMS in official OPCW proficiency test matrices.

Pinacolyl alcohol (PA), a key forensic marker for the nerve agent Soman (GD), is a particularly difficult analyte to detect by various analytical methods. In this work, we have explored the reaction between PA and 1,1'-carbonyldiimidazole (CDI) to yield pinacolyl 1H-imidazole-1-carboxylate (PIC), a product that can be conveniently detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Regarding its GC-MS profile, this new carbamate derivative of PA possesses favorable chromatographic features such as a sharp peak and a longer retention time (RT = 16.62 min) relative to PA (broad peak and short retention time, RT = 4.1 min). The derivative can also be detected by LC-HRMS, providing an avenue for the analysis of this chemical using this technique where PA is virtually undetectable unless present in large concentrations. From a forensic science standpoint, detection of this low molecular weight alcohol signals the past or latent presence of the nerve agent Soman (GD) in a given matrix (i.e., environmental or biological). The efficiency of the protocol was tested separately in the analysis and detection of PA by EI-GC-MS and LC-HRMS when present at a 10 μg/mL in a soil matrix featured in the 44th PT and in a glycerol-rich liquid matrix featured in the 48th Official Organization for the Prohibition of Chemical Weapons (OPCW) Proficiency Test when present at a 5 μg/mL concentration. In both scenarios, PA was successfully transformed into PIC, establishing the protocol as an additional tool for the analysis of this unnatural and unique nerve agent marker by GC-MS and LC-HRMS.

LC-MS/MS-QTOF Screening and Identification of Phenolic compounds from Ethyl Acetate Fraction of Madhuca longifolia Leaves

Natural products, particularly those derived from plants and microbes, offer an endless supply of unique compounds that can be converted into new pharmaceuticals. Due to their potential health benefits, bioactive components from herbs, particularly phenolics, have recently attracted a lot of interest. One of the multi-use forest tree species, Madhuca longifoliaJ. Koenig. (mahua), is found throughout much of South Asia and offers locals a source of food, fuel, fodder, and other non-timber forest products (NTFPs) in addition to timber. Almost all of this tree's components exhibit its therapeutic qualities.Mahua's phytochemical analysis reveals that it contains high levels of vitamins, alkaloids, sugar, glycosides, proteins, steroids, tannins, flavonoids, saponins, terpenoids, and phenolic compounds. These compounds are responsible for a variety of pharmacological properties, including anti-inflammatory, antioxidant, analgesic, antihyperglycemic, spasmolytic, hepatoprotective, anticonvulsant, anticancer, results of the High-Resolution Liquid Chromatography Mass Spectroscopy (HR-LCMS) analysis showed that plant extracts included a number of pharmaceutically significant chemicals. By comparing the weights and fragmentation patterns of the twenty-four (24) phenolic compounds in this work with those of published libraries, the LC-MS/MS method was used to screen and confirm them. The findings of this research support the usage of this plant as a source of bioactives and the beneficial effects of these compounds on human health.

Synthetic cathinones in drug-facilitated sexual assault: A case report involving the novel generation substituted cathinone N-ethylpentedrone and a review of the literature

The use of 3,4-methylenedioxymethamphetamine (MDMA) in drug-facilitated sexual assault (DFSA) is not uncommon. Indeed, the effects associated with the use of this substance may lead to disinhibition. Several synthetic cathinones, such as mephedrone or methylone, also possess marked entactogenic properties. This manuscript aims to (i) report a DFSA case involving a novel cathinone derivative, namely N-ethyl-pentedrone (NEPD) and (ii) review previously reported DFSA cases involving synthetic cathinones. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), NEPD was detected in both plasma and urine collected from a 36-year-old male who had been victim of DFSA. Furthermore, an exhaustive, non-period-specific English-language literature search was performed using several different electronic databases to identify DFSA cases involving synthetic cathinones. Overall, five synthetic cathinones have been associated with DFSA:methylenedioxypyrovalerone, 4-methylethcathinone, α -pyrrolidinopentiophenone, mephedrone, α –pyrrolidinohexiophenone, and methylone, which appears to be the most frequently reported. Methylone is the β-keto analog of MDMA, with which it shares substantial pharmacological similarities. Indeed, the pharmacological effects of methylone are similar to those associated with MDMA. By contrast, little is known regarding NEPD’s pharmacological effects in humans. Based on subjective reports, NEPD can produce both positive and negative effects in human. Unlike what is reported in the case of methylone or mephedrone, only a small minority of NEPD users report slightly entactogenics effects. Such properties theoretically make NEPD more suitable for use in a chemsex context than in DFSA context; even though, the boundary between these two specific forms of sexualized drug use can sometimes appear tenuous.

Japanese Planocerid Flatworms: Difference in Composition of Tetrodotoxin and Its Analogs and the Effects of Ingestion by Toxin-Bearing Fishes in the Ryukyu Islands, Japan

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography–mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

Fragmentation characteristics-based nontargeted screening method of exogenous chemical residues in animal-derived foods using reversed-phase and hydrophilic interaction liquid chromatography–high-resolution mass spectrometry

Fragmentation characteristics are crucial for nontargeted screening to discover and identify unknown exogenous chemical residues in animal-derived foods. In this study, first, fragmentation characteristics of 51 classes of exogenous chemical residues were summarized based on experimental mass spectra of standards in reversed-phase and hydrophilic interaction liquid chromatography–high-resolution mass spectrometry (MS) and mass spectra from the MassBank of North America (MoNA) library. According to the proportion of fragmentation characteristics to the total number of chemical residues in each class, four screening levels were defined to classify 51 classes of chemical residues. Then, a nontargeted screening method was developed based on the fragmentation characteristics. The evaluation results of 82 standards indicated that more than 90 % of the chemical residues with MS/MS spectra can be identified at concentrations of 100 and 500 μg/kg, and about 80 % can be identified at 10 μg/kg. Finally, the nontargeted screening method was applied to 16 meat samples and 21 egg samples as examples. As a result, eight chemical residues and transformation products (TPs) of 5 classes in the exemplary samples were found and identified, in which 3 TPs of azithromycin were identified by fragmentation characteristics-assisted structure interpretation. The results demonstrated the practicability of the nontargeted screening method for routine risk screening of food safety.

Development and validation of high-performance liquid chromatography-Orbitrap mass spectrometric method for quantification of NDMA in ranitidine drug products and evaluation of antioxidants as inhibitors of classical nitrosation reaction.

N-Nitroso dimethylamine (NDMA) is a mutagenic impurity detected in several ranitidine products. The amino functional group of ranitidine is a risk factor for classical nitrosation-induced NDMA formation in ranitidine drug products during storage conditions. The United States Food and Drug Administration (US FDA) recommended the use of antioxidants to control NDMA in drug products. Considering the need for sensitive analytics, a liquid chromatography/high-resolution mass spectrometry (LC-HRMS) method was developed and validated to detect NDMA in this pilot study to demonstrate the antioxidants as inhibitors of nitrosation reactions. The method, utilizing an EC-C18 column and tuned to atmospheric pressure chemical ionization/selected ion monitoring (APCI/SIM) mode, separated NDMA (m/z: 75.0553; tR: 3.71 min) and ranitidine (m/z: 315.1485; tR: 8.61 min). APCI mode exhibited four times higher sensitivity to NDMA than electrospray ionization (ESI) mode. Classical nitrosation of the dimethyl amino group of ranitidine was studied with sodium nitrite in solid pellets. Antioxidants (alpha-tocopherol, ascorbic acid, and trolox) were evaluated as NDMA attenuators in ranitidine pellets under vulnerable storage conditions. The developed method quantified NDMA levels in samples, extracted with methanol through vortex shaking for 45 min. The method achieved a limit of detection (LOD) and limit of quantitation (LOQ) of 0.01 and 0.05 ng/mL, respectively, with linearity within 1-5000 ng/mL (R1: 0.9995). It demonstrated good intra-day and inter-day precision (% RSD [relative standard deviation]: <2) and accuracy (96.83%-101.72%). Nitrosation of ranitidine induced by nitrite was significant (p < 0.001; R2 = 0.9579) at various sodium nitrite levels. All antioxidants efficiently attenuated NDMA formation during ranitidine nitrosation. Ascorbic acid exhibited the highest NDMA attenuation (96.98%), followed by trolox (90.58%). This study recommends 1% ascorbic acid and trolox as potent NDMA attenuators in ranitidine drug products. This study compared the effectiveness of antioxidants as NDMA attenuators in ranitidine under storage conditions susceptible to NDMA generation. The study concluded that ascorbic acid and trolox are potent inhibitors of NDMA formation and nitrosation attenuators in ranitidine drug products.

Identification of hot spots and co-occurrence patterns of activities on thyroid hormone receptor and transthyretin binding in passive samplers from Czech surface waters

One of the less studied in vitro biological activities in the aquatic environment are thyroid hormone receptor beta (TRβ)-mediated agonistic and antagonistic activities and transthyretin (TTR) binding activity. They were measured mostly using active sampling methods, but rarely found. It is unclear if these activities co-occur, and the drivers of the (anti-)TRβ activity are mostly unknown. Therefore, the main aim of the study was to determine (anti-)TRβ activities as well as transthyretin (TTR) binding activity in passive samplers from Czech surface waters in combination with the search for the effect drivers based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis by applying suspect screening. Passive samplers (polar organic chemical integrative samplers, POCIS) were deployed at twenty-one sites (all ends of watersheds and other important sites in Elbe River) in the Czech rivers. The (anti-)TRβ and TTR binding activity were measured using (anti-)TRβ-CALUX and TTR-TRβ-CALUX bioassays. Anti-TRβ activity was found at eight sites, and TTR binding activity co-occurred there at six of these sites. The co-occurrence of TRβ-mediated antagonistic activity and TTR binding indicate that they may have common effect drivers. No sample exhibited TRβ agonistic activity. The extract from the site Bílina River, the most burdened with anti-TRβ activity, was further successfully fractionated, and this activity was revealed in the fraction, where mid-polar compounds prevailed. However, the suspect LC-HRMS analysis did not reveal the chemical effect drivers. Our results showed that anti-TRβ activity can be found in surface waters by employing passive sampling and frequently co-occurs with TTR binding activity. Overall, the fractionation procedure and non-target data acquisition used in this study can serve as a basis for searching the effect drivers in future research.