Liquid Chromatography-high Resolution Mass Spectrometry Analysis Research Articles

LC-HRMS analysis of phospholipids bearing oxylipins

BackgroundSeveral oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids act as lipid mediators. In biological samples they can be present as non-esterified form, but the major part occurs esterified in phospholipids (PL) or other lipids. Esterified oxylipins are quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. However, in this indirect analysis the information in which lipid class oxylipins are bound is lost. In this work, an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of PL bearing oxylipins was developed. ResultsOptimized reversed-phase LC separation achieved a sufficient separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers. Individual PL species bearing oxylipins were identified based on retention time, precursor ion and characteristic product ions. The bound oxylipin could be characterized based on product ions resulting from the α-cleavage occurring at the hydroxy/epoxy group. PL sn-1/sn-2 isomers were identified based on the neutral loss of the fatty acyl in the sn-2 position. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC-P, PC-O, and PE-P were detected in human serum and cells. This method enabled to determine in which PL class supplemented oxylipins are incorporated in HEK293 cells: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other oxylipins were mainly found in PC. SignificanceThe developed LC-HRMS method enables the comprehensive detection as well as the semi-quantification of isobaric and isomeric PL species bearing oxylipins. With this method, we show that the position of the oxidation has a great impact and directs the incorporation of oxylipins into the different PL classes in human cells.

Enhanced N-Glycan Profiling of Therapeutic Monoclonal Antibodies through the Application of Upper-Hinge Middle-Up Level LC-HRMS Analysis.

Therapeutic monoclonal antibodies (mAbs) are crucial in modern medicine due to their effectiveness in treating various diseases. However, the structural complexity of mAbs, particularly their glycosylation patterns, presents challenges for quality control and biosimilarity assessment. This study explores the use of upper-hinge middle-up (UHMU)-level ultra-high-performance liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis to improve N-glycan profiling of mAbs. Two specific enzymes, known as IgG degradation enzymes (IGDEs), were used to selectively cleave therapeutic mAbs above the hinge region to separate antibody subunits for further Fc glycan analysis by means of the UHMU/LC-HRMS workflow. The complexity of the mass spectra of IGDEs-digested mAbs was significantly reduced compared to the intact MS level, enabling reliable assignment and relative quantitation of paired Fc glycoforms. The results of the UHMU/LC-HRMS analysis of nine approved therapeutics highlight the significance of this approach for in-depth glycoform profiling.

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.

Multiplatform Metabolomics to Understand the Imidacloprid-Induced Toxicity in Drosophila.

Neonicotinoids, the class of insecticides used for crop protection, are subjected to vigilance due to their pernicious impacts. Imidacloprid (IMD) is one of the most representative insecticides of the neonicotinoid family, which has shown unfriendly consequences for non-target species. Metabolomics, a multidisciplinary approach, is being used in toxicological research to understand the metabolic responses to toxicant exposure by utilizing modern analytical techniques. Yet, no solitary analytical technique can cover the broad metabolite spectrum, but a multi-technique metabolomics platform can aid in analyzing the majority of the metabolites. In the present study, an effort has been made to identify the differential metabolites in Drosophila after exposure to IMD at 2.5 and 25 ng/mL using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), gas chromatography-MS (GC-MS), and NMR-based untargeted metabolomics. Multivariate pattern recognition analysis helped in identifying/recognizing 19 (LC-HRMS), 7 (GC-MS), and 13 (NMR) differential metabolites mainly belonging to the category of amino acids, sugars, fatty acids, and organic acids. The pathway analysis of differential metabolites predominantly showed impact on aminoacyl-tRNA biosynthesis, amino acid metabolism, and glycerophospholipid metabolism. Among these, arginine and proline metabolism was observed to be the common metabolic pathway perturbed in Drosophila due to IMD exposure. The multiplatform metabolomics based on LC-HRMS, GC-MS, and NMR analysis with an advanced level of statistical analysis can provide insights into potential perturbations in the metabolome of IMD-exposed Drosophila.

LC-HRMS and FTIR-based metabolomics analysis and xanthine oxidase inhibitory evaluation of Sida rhombifolia with different drying methods

Sida rhombifolia, known as Sidaguri in Indonesia, has been widely used in traditional medicine because of its bioactive metabolites, particularly flavonoids and steroids. Several factors, including the drying method, can affect the composition and concentrations of metabolites in plants. This study aimed to profile the metabolites and evaluate the xanthine oxidase inhibitory activity of S. rhombifolia dried by different methods. Three drying methods were used: air drying (AD), oven drying (OD), and sun drying (SD). Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and Fourier transform infrared (FTIR)-based untargeted metabolomics were used to profile the metabolites in S. rhombifolia. The FTIR spectra of samples dried by different methods showed similar patterns, with peaks at 3398, 2935, 2856, 1728, 1456, and 1164 cm−1. Metabolite profiling of S. rhombifolia using liquid chromatography–tandem mass spectrometry (LC–MS/MS) identified 24 putative metabolites, with 20, 15, and 16 metabolites found in the AD, OD, and SD extracts, respectively. The principal component analysis demonstrated the separation of S. rhombifolia into groups based on drying method differences. These differences account for 95% (LC–MS/MS) and 99% (FTIR) of the total variance explained. Evaluation of xanthine oxidase inhibition revealed that S. rhombifolia inhibited the enzyme by 22.81% (AD), 35.97% (OD), and 45.93% (SD) at an extract concentration of 50 ppm. The drying method affected the metabolite profile with regard to both the functional groups and amounts of metabolites. Furthermore, the results showed that the xanthine oxidase inhibitory activity depended on the S. rhombifolia drying method and conditions.

Aqueous-phase photo-oxidation of selected green leaf volatiles initiated by [rad]OH radicals: Products and atmospheric implications

C5- and C6- unsaturated oxygenated organic compounds emitted by plants under stress like cutting, freezing or drying, known as Green Leaf Volatiles (GLVs), may clear some of the existing uncertainties in secondary organic aerosol (SOA) budget. The transformations of GLVs are a potential source of SOA components through photo-oxidation processes occurring in the atmospheric aqueous phase. Here, we investigated the aqueous photo-oxidation products from three abundant GLVs (1-penten-3-ol, (Z)-2-hexen-1-ol, and (E)-2-hexen-1-al) induced by OH radicals, carried out in a photo-reactor under simulated solar conditions. The aqueous reaction samples were analyzed using advanced hyphenated mass spectrometry techniques: capillary gas chromatography mass spectrometry (c-GC–MS); and reversed-phase liquid chromatography high resolution mass spectrometry (LC-HRMS). Using carbonyl-targeted c-GC–MS analysis, we confirmed the presence of propionaldehyde, butyraldehyde, 1-penten-3-one, and 2-hexen-1-al in the reaction samples. The LC-HRMS analysis confirmed the presence of a new carbonyl product with the molecular formula C6H10O2, which probably bears the hydroxyhexenal or hydroxyhexenone structure. Density functional theory (DFT)-based quantum calculations were used to evaluate the experimental data and obtain insight into the formation mechanism and structures of the identified oxidation products via the addition and hydrogen-abstraction pathways. DFT calculations highlighted the importance of the hydrogen abstraction pathway leading to the new product C6H10O2. Atmospheric relevance of the identified products was evaluated using a set of physical property data like Henry's law constant (HLC) and vapor pressure (VP). The unknown product of molecular formula C6H10O2 has higher HLC and lower VP than the parent GLV and thus has potential to remain in the aqueous phase leading to possible aqueous SOA formation. Other observed carbonyl products are likely first stage oxidation products and precursors of aged SOA.

In vitro tyrosinase inhibitory, DNA interaction studies, and LC-HRMS analysis of Ficus carica leaves.

Turkey is the world's leading producer of figs, a typical Mediterranean fruit. The fig, Ficus carica L. (Moraceae), has been widely cultivated since ancient times due to the nutritional value of its fruits. It was aimed to investigate the phytochemical characterization and biological properties of F. carica leaf extracts in order to determine their potential for use in the treatment of various diseases. F. carica leaves were extracted in 70% methanol at 40 °C under reflux. To obtain extracts of different polarities, the crude extract was fractionated with n-hexane, dichloromethane, and n-butanol. Phenolic content was determined using liquid chromatography-high resolution mass spectrometry (LC-HRMS). 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and antityrosinase activities of all extracts were investigated using spectrophotometric methods. Furthermore, the DNA-damage protective properties of extracts were investigated using electrophoretic methods. The n-butanol extract was found to have the highest total phenolic content, with 72.58 ± 4.52 mg GAE/g dry weight. According to LC-HRMS analysis, rutin (40.13 g/kg) was the most abundant compound in the n-butanol extract. The n-butanol extract, which was found to have the highest tyrosinase inhibitory effects among the extracts, demonstrated radical scavenging activity of 37.01 ± 1.15% and 82.57 ± 0.88% at 80 and 200 μg/mL, respectively. The n-butanol extract had the highest protective effects against Fenton's reagent, UV radiation, and singlet oxygen. Given these findings, it is possible to argue that F. carica leaves can be evaluated for developing products that could be used to treat various diseases.

Glucosinolates and Polyphenols of Colored Cauliflower as Chemical Discriminants Based on Cooking Procedures.

The impact of mild oven treatments (steaming or sous-vide) and boiling for 10 min, 25 min, or 40 min on health-promoting phytochemicals in orange and violet cauliflower (Brassica oleracea L. var. botrytis) was investigated. For this purpose, targeted ultra-high performance liquid chromatography–high-resolution mass spectrometry analysis of phenolics and glycosylates, combined with chemometrics, was employed. Regardless of cooking time, clear differentiation of cooked samples obtained using different procedures was achieved, thus demonstrating the distinct impact of cooking approaches on sample phytochemical profile (both, compound distribution and content). The main responsible components for the observed discrimination were derivatives of hydroxycinnamic acid and kaempferol, organic acids, indolic, and aromatic glucosinolates, with glucosativin that was found, for the first time, as a discriminant chemical descriptor in colored cauliflower submitted to steaming and sous-vide. The obtained findings also highlighted a strict relationship between the impact of the cooking technique used and the type of cauliflower. The boiling process significantly affected the phytochemicals in violet cauliflower whereas orange cauliflower boiled samples were grouped between raw and either steamed or sous-vide-cooked samples. Finally, the results confirm that the proposed methodology is capable of discriminating cauliflower samples based on their phytochemical profiles and identifying the cooking procedure able to preserve bioactive constituents.

Comparison of untargeted chemical profiles of dried blood spots, dried blood microsamplers, and plasma

Background and aim: Capillary blood collection is minimally invasive, easy to perform, and when dried, is more stable at higher temperatures than liquid blood, making dried capillary blood microsamplers a promising alternative to venous blood for epidemiological research. To evaluate the utility of dried blood samplers for use in exposome studies, comparisons of metabolite profiles between capillary dried blood and conventional plasma collection are needed. Methods: Commercially purchased whole blood from a single adult smoker was used for experiments. Dried blood spots were generated by pipetting 50 µL for 5-mm punches, 10 µL VAMS were generated from the whole blood following manufacturer’s instructions, and 10 µL plasma aliquots were generated by centrifugation. Samples were stored overnight at -80C. Triplicate samples were randomized and analyzed for each device using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). Metabolites were identified using Agilent Profinder software. Metabolite levels (abundance) were compared for each sample type using a one-way ANOVA and fold change (FC). Results: We identified 305 metabolites from LC-HRMS analysis. Among those compounds, 222 metabolites show significant differences in abundance among Guthrie, VAMS, and plasma samples (p value<0.05). 42 compounds have higher abundance (>3 FC) in Guthrie or VAMS than plasma samples. Among these compounds, carboxylic acids and derivatives (7) and organooxygen compounds (7) are the major compound classes, but also included two metabolites of alcohol exposure. Three compounds have a higher abundance (>3 FC) in plasma than Guthrie or VAMS. Conclusions: Several metabolites have a higher abundance in Guthrie or VAMS than in plasma samples. However, most compounds showed a comparable fold change among these three sample types. Depending on the group of metabolites or exogenous compounds of interest, microsampling devices are an alternative to conventional plasma collection for exposome studies. Keywords: Untargeted chemical analysis, dried blood spots, microsamplers, exposome.

Unfolding Biomechanism of Dolichos lablab Bean as A Dietary Supplement in Type 2 Diabetes Mellitus Management through Computational Simulation

Dietary intervention, particularly legumes consumption, plays a significant role in promoting health status in diabetes mellitus management. As poorly known legumes, Dolichos lablab (DL) is possibly to be one of the dietary options for diabetes intervention. However, the predictive or precise mechanism of DL’s anti-diabetic activity remains inconclusive. This study aimed to determine the nutritional and phytochemical content in addition to anti-diabetic properties of DL. Total protein, crude fat, crude fibers, and gross energy were evaluated, while anti-diabetic properties were predicted using molecular docking according to identified compound from Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) analysis. Screened compound from molecular docking then passed to physicochemical properties and bioactivity prediction using Swiss-ADME and molinspiration, separately. The result showed that DL has high protein fiber and gross energy content with a lower fat percentage. Additionally, DL has numerous phenolic acid and flavonoid compounds according to LC-HRMS analysis. From the docking analysis, fourteen compounds have substantial probability to give the beneficial effect of glucose metabolism regulator and insulin signaling repairers through inhibition of ɑ-amylase, DPP4, and PTP1B. Finally, from the physicochemical properties and bioactivity estimations, 19-Norandrostenedione, 19-Nortestosterone, Icariside B1, Ilicic Acid, and Psilostachyin B have excellent pharmacokinetic properties along with considerable biological activity as enzyme inhibitors and nuclear receptor ligands. In conclusion, nutritional evaluation and molecular docking analysis revealed that DL might serve as a suitable dietary intervention for diabetes mellitus management.

The Role of Ipomoea batatas Leaves Extract as a Potent Antioxidant and Anti-inflammatory in Rats Fed High-fat Diet

Ipomoea batatas L purple variant is a natural source of bioactive compounds with strong antioxidant activity and has long been used in folk medicine. This study aimed to evaluate the active compounds, antioxidant, and anti-inflammatory activities of Ipomoea batatas L purple variant leaf extract in rats fed a high-fat diet. The presence of active compounds in the Ipomoea batatas L purple variant was determined by liquid chromatography high resolution mass spectrometry (LC-HRMS). The rats were divided into five experimental groups of six animals in each group. Namely standard diet/normal group (SD), high-fat diet group (HFD), and HFD plus extracts (625; 1250; or 2500 mg/kg) groups for twelve weeks. The effect of leaf extract on antioxidant activity was analyzed using the DPPH assay, while oxidative stress and anti-inflammatory analysis were performed by immunohistochemistry. LC-HRMS analysis showed six active compounds were identified from Ipomoea batatas L. Besides, there is a reduction in oxidative stress through malondialdehyde (MDA) expression on the oral supplementation with Ipomoea batatas leaf purple variant extract doses of 1250 and 2500 mg/kg body weight. Meanwhile, all extract doses can significantly increase antioxidant activities such as nuclear factor-erythroid 2 related factor 2 (Nrf-2) expression, superoxide dismutase 2 (SOD2) expression, and decrease pro-inflammatory such as tumor necrosis factor alpha (TNF-α) expression in the aorta. Taken together, these findings provide a basis for the recommendation of compounds from the supplementation of an Ipomoea batatas L purple variant leaf extract can be an alternative herbal therapy to inhibit the harmful effects of a high-fat diet.

Screening and Toxicity Assay of Bioactive Compounds from Ethyl Acetate Extract of Indonesian Marine Sponge <i>Dysidia </i>sp<i>.</i> from Hoga Island

Screening of the toxic active compounds from the ethyl acetate fraction of the sponge Dysidea sp. from Hoga Island, Wakatobi National Park. This study aims to determine the toxic properties of the ethyl acetate fraction of sponge Dysidea sp. Isolation was carried out by extraction method using methanol as solvent, then the extract was partitioned using various solvents, such as N-Hexane, Dichloromethane, Ethyl Acetate and Acetone. The obtained fraction was then tested for toxicity using the Brine Shrimp Lethality Test (BSLT) and phytochemical tests. Identification of compounds from the fraction that have toxic properties were then identified using Liquid Chromatography – High Resolution Mass Spectrometry (LC-HRMS). The ethyl acetate fraction obtained was 1.5 grams in the form of a green solid paste. LC-HRMS analysis showed that the ethyl acetate fraction contained 3 reported compounds and 2 unreported compounds.

In Vitro Antilipidic and Antithrombotic Activities of Plectranthus glandulosus (Lamiaceae) Leaves Extracts and Fractions.

Objective The present study investigated the effect of the leaves extracts and fractions of Plectranthus glandulosus on the inhibition of pancreatic lipase, cholesterol esterase, adipocytes lipid uptake, and antithrombotic activity which may be important in atherosclerosis development. Methods Aqueous, ethanolic, and hydroethanolic extracts of Plactranthus glandulosus were prepared by maceration. The hydroethanolic extract was fractionated into n-hexane, ethylacetate, and n-butanol fractions and their inhibition of pancreatic lipase, cholesterol esterase, adipocytes lipid uptake, and antithrombotic activities measured. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis was carried out to determine phytochemical constituents present in the extracts. Results The standard orlistat exhibited a higher inhibitory activity on pancreatic lipase and cholesterol esterase (16.31 μg/mL and 15.75 μg/mL, respectively) compared to ethyl acetate fraction (IC50, 17.70 μg/mL and IC50, 24.8 μg/mL, respectively). Among crude extract, hydroethanolic extract showed a better inhibition against pancreatic lipase (IC50, 21.06 μg/mL) and cholesterol esterase (IC50, 25.14 μg/mL) though not comparable to the effect of orlistat. The best lipid uptake inhibition was observed in the hydroethanolic extract (IC50, 45.42 μg/mL) followed by the ethyl acetate fraction (IC50, 47.77 μg/mL). A better antithrombolytic activity was exhibited by the ethyl acetate fraction at all concentrations (50-800 μ/mL), while hydroethanolic extract exhibited the best activity among crude extract. However, these were not comparable to the standard aspirin. The LC-HRMS analysis revealed the presence of 7-O-methyl luteolin 5-O-β-D-glucopyranoside, chrysoeriol 5-O-β-D-glucopyranoside, 5,7-dihydroxy-3,2′,4′-trimethoxyflavone, and plectranmicin as major compounds in both hydroethanolic extract and ethyl acetate fraction. Conclusion Thus, our finding supports the traditional use of this plant, which might provide a potential source for future antiatherosclerotic drug discovery.

24 Lipid composition of fresh or frozen sexed bovine blastocysts produced invivo or invitro

Currently, invitro embryo production (IVP) is successfully applied commercially in cattle. However, the high sensitivity of embryos to cryopreservation compared with invivo-derived (IVD) embryos still impairs the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to invitro culture conditions. The objective of this study was to evaluate the lipid content of fresh and frozen sexed bovine grade 1 IVP or IVD embryos. The same 8 Holstein heifers were used in a Latin square design for both IVP and IVD embryo production. Zygotes were cultured in synthetic oviductal fluid (SOF) supplemented with 1% oestrus cow serum. The same bull was used for IVP and IVD. All expanded Day 7 blastocysts (n=40 IVP and 40 IVD) were biopsied and sexed. Half of the embryos (n=20 in each group) was slow frozen (1.5M ethylene glycol, 0.1m sucrose) and thawed before lipid extraction. Remaining embryos underwent lipid extraction in the fresh state. Briefly, the liposoluble fraction of the embryos was extracted according to the Bligh and Dyer method using chloroform and methanol. Liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis was performed and operated in positive ionization mode. Lipids with variance intensities greater than 30% in quality control samples were removed as well as those identified as background noise. Partial least square discriminant analysis (PLS-DA) was used to show the relationship between variance in the data and difference among embryo origin (IVP vs. IVD), state before extraction (fresh vs. frozen), and sex of the embryos (male vs. female). The differentially lipid species groups were identified using Wilcoxon test, and considered significantly different when P<0.05. LC-HRMS analysis allowed us to identify 75 lipids. PLS-DA showed that embryo origin (IVP vs. IVD) and state before extraction (fresh vs. frozen) can be determined by LC-HRMS profiles by group in PLS-DA plot, despite slight overlaps. Sex of the embryos did not allow us to differentiate the lipid profile. However, 15 lipids varied significantly between male and female IVD, predominantly triglycerides (TG), whereas no lipid varied between the sexes in the IVP homologues. Moreover, 26 lipids varied significantly between IVP and IVD fresh embryos with enrichment of IVP embryos in TG, phosphatidyl choline, cholesteryl ester, and less diglyceride and lysophospholipid (LP) compared with IVD embryos. The comparison of the lipid profiles before and after freezing for IVP embryos showed that only 7 lipids varied significantly between fresh and frozen states with a decrease in LP for the frozen embryos. For the invivo counterparts, 13 lipids varied significantly, including the same LP as those identified for IVP embryos in the same way. Our results showed that the embryonic lipid profile is mainly affected by IVP and slow freezing protocols and, to a lesser extent, by sex. Further studies are needed to improve IVP protocols and optimize the cryotolerance of IVP embryos in cattle.

Fluorescence polarisation for high‐throughput screening of adulterated food products via phosphodiesterase 5 inhibition assay

The surge in the consumption of food products containing herbal aphrodisiacs has driven their widespread adulteration. A rapid screening strategy is, therefore, warranted to curb this problem. This study established an enzyme inhibition assay to screen phosphodiesterase 5 (PDE5) inhibitors as adulterants in selected food products. Fluorescein-labelled cyclic-3',5'-guanosine monophosphate was utilised as substrates for the PDE5A1 enzyme, aided by the presence of nanoparticle phosphate-binding beads on their fluorescence polarisation. The sample preparation was optimised to improve the enzyme inhibition efficiency and applied to calculate the threshold values of six blank food matrices. The assay was validated using sildenafil, producing an IC50 of 4.2nM. The applicability of the assay procedure was demonstrated by screening 55 distinct food samples. The results were subsequently verified using confirmatory liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis. Altogether, 49 samples inhibited the PDE5 enzyme above the threshold values (75.7%-105.5%) and were registered as potentially adulterated samples. The remaining six samples were marked as nonadulterated with percentage inhibition below the threshold values (-3.3%-18.2%). The LC-HRMS analysis agreed with the assay results for all food products except for the instant coffee premix (ICP) samples. False-positive results were obtained for the ICP samples at 32% (8/25), due to possible PDE5 inhibition by caffeine. Contrarily, all other food samples were found to produce 0% (0/30) false-positive or false-negative results. The broad-based assay, established via a simple mix-incubate-read format, exhibited promising potential for high-throughput screening of PDE5 inhibitors in various food products, except those with naturally occurring phosphodiesterase inhibitors such as caffeine.

Comparison of Three Untargeted Data Processing Workflows for Evaluating LC-HRMS Metabolomics Data.

The evaluation of liquid chromatography high-resolution mass spectrometry (LC-HRMS) raw data is a crucial step in untargeted metabolomics studies to minimize false positive findings. A variety of commercial or open source software solutions are available for such data processing. This study aims to compare three different data processing workflows (Compound Discoverer 3.1, XCMS Online combined with MetaboAnalyst 4.0, and a manually programmed tool using R) to investigate LC-HRMS data of an untargeted metabolomics study. Simple but highly standardized datasets for evaluation were prepared by incubating pHLM (pooled human liver microsomes) with the synthetic cannabinoid A-CHMINACA. LC-HRMS analysis was performed using normal- and reversed-phase chromatography followed by full scan MS in positive and negative mode. MS/MS spectra of significant features were subsequently recorded in a separate run. The outcome of each workflow was evaluated by its number of significant features, peak shape quality, and the results of the multivariate statistics. Compound Discoverer as an all-in-one solution is characterized by its ease of use and seems, therefore, suitable for simple and small metabolomic studies. The two open source solutions allowed extensive customization but particularly, in the case of R, made advanced programming skills necessary. Nevertheless, both provided high flexibility and may be suitable for more complex studies and questions.

Determination of estrogens by solid-phase quadruplex stable isotope dansylation coupled with liquid chromatography-high resolution mass spectrometry in environmental samples

Estrogens distribute widely in the environment as endocrine-disrupting chemicals (EDCs), which have to be monitored to evaluate their environmental impact. Aim to improve the analytical throughput of liquid chromatography-high resolution mass spectrometry (LC-HRMS), a quadruplex stable isotope dansylation method was developed, with which three real samples could be quantitatively analyzed in one injection. As the estrogens were at trace level in complex matrices, magnetic solid-phase extraction (MSPE) was applied to enrich these analytes and remove the interfaces. By integrating MSPE and quadruplex stable isotope dansylation, a solid-phase quadruplex labeling method was developed for the LC-HRMS analysis of estrogen analogues. For the tested seven estrogens, the developed method showed low detection limits (0.1–0.5 ng/L for pond water and 0.01–0.05 μg/kg for poultry manure), good precision (RSD < 5.5%) and accuracy (96.8–108.3%). The method was applied in the determination of estrogens in environmental samples, and the results revealed that all the tested estrogens were present in the estuary water (unquantifiable to 71.2 ng/L) and chicken manure (undetectable to 25.43 μg/kg).

Characterization and identification of charcoal of inedible Kerandang fish (Channa pleurophthalmus Blkr) body parts and potential antiallergenic properties.

Background and Aim:The study about the antiallergenic properties of inedible fish body parts is still limited. Therefore, this study aimed to characterize the charcoal from the body parts of Kerandang fish (Channa pleurophthalmus Blkr) and identify its antiallergenic properties.Materials and Methods:This study used some non-edible body parts extracted from the Kerandang fish (i.e., the scalp, scales, and dorsal, pectoral, ventral, anal, and caudal fins) using a maceration method with different solvents (ethanol, ethyl acetate, and chloroform). The identification of active compounds in the extract was carried out using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis, while the antihyaluronidase activity was determined using the antihyaluronidase test. The highest charcoal antihyaluronidase activity-extract was applied to ovalbumin-induced mice for 7 days with various doses (10, 15, and 20 mg/kg). The specific immunoglobulin E (IgE) was measured using enzyme-linked immunosorbent assay on day 8.Results:Our LC-HRMS analysis showed that the active compound of charcoal in the caudal fins of Kerandang fish was hexadecanamide. The highest inhibition (IC50) of hyaluronidase was found in the ethyl acetate extract of fish caudal fins at a concentration of 4 mg/mL. We found that 15 mg/kg body weight of charcoal of fish caudal fins suppressed IgE expression in male mice.Conclusion:Our findings indicate that the charcoal of non-edible body parts of Kerandang and one of its constituent, hexadecanamide, may have strong antiallergic effects.

Chemical Composition, Toxicity, Antinociceptive, and Anti-Inflammatory Activity of Dry Aqueous Extract of Varronia multispicata (Cham.) Borhidi (Cordiaceae) Leaves.

Varronia multispicata (Cham.) Borhidi (Cordiaceae), an herbaceous plant distributed in tropical and subtropical regions is native of Brazil and widely used in folk medicine to treat respiratory and digestive diseases, inflammation, and some types of infections. Thus, this study aimed to investigate acute oral toxicity, antinociceptive, and anti-inflammatory activities of dry aqueous extract of V. multispicata (AEVm) and to identify its compounds. Extract was obtained by lyophilized leaf infusion and its composition was analyzed by ultra-performance liquid chromatography-high resolution mass spectrometry (LC-MS). Acute oral toxicity was evaluated in female rats treated with AEVm (2,000 mg/kg) in a single oral dose. Mortality, body weight changes, feed and water intake, organ weights, histological and biochemical parameters were screened for 14 days. Antinociceptive activity was evaluated by writhing (WT), formalin (FT), and hot plate (HP) tests in male mice while anti-inflammatory activity was performed by carrageenan (CPE) and dextran (DPE)-induced paw edema tests and carrageenan-induced peritonitis (CP) test in male rats. Additionally, spontaneous open-field (OF) locomotion was evaluated. LC-MS analysis revealed the presence of flavonoids with biological activity. In toxicity evaluation, extract did not cause deaths in dose of 2,000 mg/kg, and there were no significant behavioral or biochemical alterations. Additionally, evidence of hepatoprotective and antioxidant activity was observed. In pharmacological evaluation AEVm showed dose-dependent antinociceptive activity in WT, with a median effective dose of 146.89 mg/kg, which showed selectivity by inflammatory base processes (FT first phase; p < 0.001), showing no activity in neuropathic nociception components (FT second phase and HP) or about consciousness and locomotion in OF. AEVm also showed significant anti-inflammatory activity, inhibiting CPE (p < 0.001) and cell migration (p < 0.05) and nitric oxide (NO) production (p < 0,01) in CP test. These data demonstrate that AEVm has low oral toxicity—with evidence of hepatoprotective and antioxidant properties—antinociceptive and anti-inflammatory activity, supporting V. multispicata traditional use, possibly related to flavonoids present in its constitution.

Lipid and Metabolite Profiles in Human Plasma and Associations with the Microbiome and Functional Gastrointestinal Disorders (P20-033-19)

ObjectivesMetabolomic profiling of plasma provides a biochemical fingerprint of the circulating metabolites from the host and microbiome, and may assist with understanding mechanisms underlying functional gastrointestinal (GI) disorders such as Irritable Bowel Syndrome (IBS). In a case-control study, we aimed to identify microbial and host factors in plasma to provide mechanistic insights into functional GI disorders and increase the predictability of phenotypes for use in nutrition intervention studies. MethodsPlasma samples from 246 individuals with functional GI disorders (cases) or asymptomatic (controls), consisting of 93 healthy controls, 54 with functional constipation (C) or IBS-C, 60 with functional diarrhoea (D) or IBS-D, and 39 diagnosed as IBS-mixed or awaiting diagnosis. Plasma was subjected to biphasic extraction and global metabolite profiling was performed using three separate untargeted liquid chromatography high resolution mass spectrometry analysis (LC-MS) methods (polar, semi-polar and non-polar chromatography). ResultsPlasma metabolomic profiles differed considerably between the IBS phenotypes and those of the control subjects. Multivariate partial least squares discriminant analysis of 428 annotated lipid species highlighted significant differences (P < 0.001) between IBS-C and healthy control subjects. Univariate analysis revealed significant differences in the concentrations of 48 lipid species (P < 0.05) between the two groups, including elevated concentrations of many sphingolipids and phospholipids in the IBS-C group. Biochemical network analysis also revealed major perturbations in amino acid, bile acid and lipid metabolism, and highlighted key metabolic pathways included microbial related pathways. ConclusionsPerturbations of plasma lipid concentrations in the IBS subjects suggest changes may occur with both host and microbial lipid metabolism. Future efforts to investigate these links will utilize a systems biology approach integrating metabolomic and fecal metagenomic datasets to further identify key pathways and biomarkers that characterize functional GI disorders and how nutrition can improve GI health and function. Funding SourcesFunded by the New Zealand National Science Challenge High-Value Nutrition program.