Ultra-high Performance Liquid Chromatography-mass Spectrometry Research Articles

Biocontrol Potential of Beneficial Fungus Aureobasidium pullulans Against Botrytis cinerea and Colletotrichum acutatum.

Biological control is a promising approach to reduce plant diseases caused by fungal pathogens and ensure high productivity in horticultural production. In the present study, we evaluated the biocontrol potential and underlying mechanisms of the beneficial fungus Aureobasidium pullulans against Botrytis cinerea and Colletotrichum acutatum, casual agents of gray mold and anthracnose diseases in strawberry. Notably, this is the first time that A. pullulans has been tested against C. acutatum in strawberry. A. pullulans strains (AP-30044, AP-30273, AP-53383, and AP-SLU6) showed significant variation in terms of growth and conidia production. An inverse relationship was found between the growth and conidiation rate, suggesting a trade-off between resource allocation for growth and conidial production. Dual plate co-culturing assays showed that mycelial growth of B. cinerea and C. acutatum was reduced by up to 35 and 18%, respectively, when challenged with A. pullulans compared with control treatments. Likewise, culture filtrates of A. pullulans showed varying levels of antifungal activity against B. cinerea and C. acutatum, reducing the mycelial biomass by up to 90 and 72%, respectively. Furthermore, milk powder plate assays showed that A. pullulans produced substantial amounts of extracellular proteases, which are known to degrade fungal cuticle. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analyses revealed that A. pullulans produced exophilins, liamocins, and free fatty acids known to have antifungal properties. A. pullulans shows high potential for successful biological control of strawberry diseases and discuss opportunities for further optimization of this beneficial fungus.

Bio-Assay-Guided Isolation of Fractions and Constituents with Antioxidant and Lipid-lowering Activity from Allium cepa.

Active fractions and constituents with antioxidant and lipid-lowering activities were investigated using bio-assay-guided isolation and identification. The data showed that the antioxidant fraction of A. cepa was AC50%, the main constituents of which were quercetin and isoquercitrin, by way of both ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) and bio-assay-guided purification and elucidation. Similarly, the lipid-lowering active fraction of A. cepa was AC30% with the main constituents of 3,4-dihydroxybenzoic acid and quercetin 3,4'-O-diglucoside. Also, bio-assay-guided isolation led to the isolation and identification of five known compounds with a purity of more than 98%, and quercetin was both the best free radical scavenger and lipid-lowering constituent. Moreover, the mechanism of the lipid-lowering effect of AC30% might be its reduction in mRNA expression levels of sterol regulatory element binding protein 2 (SREBP2) and FAS gene in lipid synthesis. Otherwise, reducing the mRNA expression level of lipid synthesis genes, including SREBP1, SREBP2, fatty acid synthetase (FASN), β-Hydroxy β-methylglutaryl-CoA (HMGCR), stearoyl CoA desaturase 1 (SCD1), and increasing the mRNA expression level of lipid decomposition gene, such as carnitine palmitoyl transferease-1 (CPT1), might be involved in the lipid-lowering activity of quercetin. This study suggested that Allium cepa might be used to prevent and treat oxidative stress and dislipidemia-related disorders, including NAFLD.

Membrane phospholipids and nucleotide derivatives in “big six” Escherichia coli are targets of chlorine-based sanitisers during lettuce decontamination

Sodium chlorite (NaClO), the most commonly used sanitiser for fresh produce decontamination, is associated with the problems of pungency and corrosiveness. In search for a more environmentally- and user-friendly chlorine-based sanitiser, acidic electrolysed water (AEW), came into view in recent years. To determine whether AEW is an ideal alternative to NaClO, using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), this study thoroughly investigated the effectiveness of AEW and NaClO, both containing 4 mg/L of free available chlorine (FAC), in inhibiting the “big six” Escherichia coli pathogens (serotypes O26, O45, O103, O111, O121 and O145) on lettuce as well as the underlying antimicrobial mechanisms.The two sanitisers led to generally comparable E. coli cell reductions after a 10-min treatment, which indicated the decisive role of FAC in their antimicrobial effects. Among the strains, mortalities varied from approximately 0.68 log CFU/g in O111 under both treatments to 1.48 ± 0.04 log CFU/g in AEW-treated O26 and 1.23 ± 0.04 log CFU/g in NaClO-treated O45. O111's high resistance was confirmed by its almost undisturbed metabolome whereas the high lethality of O26 and O45 could be attributed to the loss of membrane phospholipids. Besides, retarded cell growth and energy deficiency as implied by a vast depletion of nucleotide derivatives further challenged O45's survival. Overall, the comparable sanitising efficacy and the similarity in antimicrobial mechanisms between the two sanitisers provided a scientific basis for replacing NaClO with the “greener” AEW for fresh produce sanitisation in the future.

Assessment of endocrine disruptor pollutants and their metabolites in environmental water samples using a sustainable natural deep eutectic solvent-based analytical methodology

In this work, an evaluation of the occurrence of fifteen phthalates, four metabolites and one adipate in different groundwater, seawater and wastewater samples has been carried out due to their relevance on human health as they act as endocrine disruptors. For this purpose, a sustainable, fast and easy-handling vortex-assisted liquid-liquid microextraction method using a natural hydrophobic deep eutectic solvent based on menthol and carvacrol as extraction agent, combined with ultra-high performance liquid chromatography-mass spectrometry technique, has been developed and applied for the first time. An optimization was performed to evaluate four important factors affecting the extraction performance, and an analytical validation was carried out in terms of matrix effect, linearity, extraction efficiency, and sensitivity. Recovery values were obtained in the range 72–119% for all analytes (except for monoethyl phthalate: 61.1–72.3%) with relative standard deviation values lower than 17%. Limits of quantification were found between 0.91 and 8.09 μg L−1. As a result of the assessment of 31 different environmental water samples, monoethyl phthalate, diethyl phthalate, dibutyl phthalate and bis (2-ethylhexyl) phthalate were detected and quantified at different concentrations in the range 2.59–21.17 μg L−1 in 6 samples, and diallyl phthalate, butyl benzyl phthalate, dipentyl phthalate, dicyclohexyl phthalate, dihexyl phthalate and bis (2-ethylhexyl) adipate were detected in 20 more, showing the exposition of the population to these hazardous substances.

Caftaric acid oxidation in the presence of cell signaling regulator glutathione: Electrochemical and chromatographic analyses

New antioxidant materials in biomedical applications are an exciting research topic. However, the lack of understanding of the products/intermediates formed and the interaction between nucleophiles and these antioxidants will prevent commercial or industrial applications. To overcome this obstacle, chromatographic techniques, mass spectroscopy, and electrochemical analyses were combined to study the mechanism and products of the expected reaction between antioxidants and nucleophiles. Grape juice contains phenolic compounds such as 2-S-glutathionylcaftaric acid, which is formed as a result of oxidative action. Through UPLC and voltammetric analysis of caftaric acid (CFT) and its quinone with glutathione (GSH), the oxidation and subsequent reactions were monitored. Electrochemical techniques such as cyclic voltammetry (CV) and chronoamperometry (CA) have been used to investigate the electrochemical oxidation characteristics of the investigated CFT in a Britton-Robinson (B-R) buffer electrolyte. As a result of the analysis of ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) results, the oxidation products were identified to suggest the optimal mechanism for the oxidation of CFTs both in the presence and absence of GSH. A reversible oxidation process is observed at pH 1.8 for CFT without glutathione (GSH) at E1/2 = 0.52 V against Ag/AgCl 3.0 M KCl) using cyclic voltammetry. During this electrochemical process, two electrons in the CFT molecule were oxidized along with two protons, forming o-quinone. In the presence of GSH, the formed quinone either reacts with glutathione to form mono- and bi-glutathione CFT conjugates or is reduced by GSH to its original state. As a result of the UPLC-MS analysis, the oxidation products of CFT were identified, and the reaction pathways were proposed.

Metabolomics Reveals Distinctive Metabolic Profiles and Marker Compounds of Camellia (Camellia sinensis L.) Bee Pollen.

Camellia bee pollen (CBP) is a major kind of bee product which is collected by honeybees from tea tree (Camellia sinensis L.) flowers and agglutinated into pellets via oral secretion. Due to its special healthcare value, the authenticity of its botanical origin is of great interest. This study aimed at distinguishing CBP from other bee pollen, including rose, apricot, lotus, rape, and wuweizi bee pollen, based on a non-targeted metabolomics approach using ultra-high performance liquid chromatography-mass spectrometry. Among the bee pollen groups, 54 differential compounds were identified, including flavonol glycosides and flavone glycosides, catechins, amino acids, and organic acids. A clear separation between CBP and all other samples was observed in the score plots of the principal component analysis, indicating distinctive metabolic profiles of CBP. Notably, L-theanine (864.83-2204.26 mg/kg) and epicatechin gallate (94.08-401.82 mg/kg) were identified exclusively in all CBP and were proposed as marker compounds of CBP. Our study unravels the distinctive metabolic profiles of CBP and provides specific and quantified metabolite indicators for the assessment of authentic CBP.

Application of mixture analysis methods in association between metals mixture exposure and DNA oxidative damage

Objectives: To study the association between metals mixture exposure and DNA oxidative damage using mixture analysis methods, and to explore the most significant exposure factors that cause DNA oxidative damage. Methods: Workers from steel enterprises were recruited in Shandong Province. Urinary metals were measured by using the inductively coupled plasma mass spectrometry method. The level of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) was determined by using the ultra-high performance liquid chromatography-mass spectrometry method. Bayesian kernel machine regression (BKMR), elastic net regression and quantile g-computation regression were used to analyze the association between urinary metals and urinary 8-OHdG. Results: A total of 768 subjects aged (36.15±7.40) years old were included in the study. BKMR, elastic net regression and quantile g-computation all revealed an overall positive association between the mixture concentration and increased urinary 8-OHdG. The quantile g-computation results showed that with a 25% increase in metal mixtures, the urinary 8-OHdG level increased by 77.60%. The elastic net regression showed that with a 25% increase in exposure risk score, the urinary 8-OHdG level increased by 26%. The BKMR summarized the contribution of individual exposures to the response, and selenium, zinc, and nickel were significant contributors to the urinary 8-OHdG elevation. Conclusion: Exposure to mixed metals causes elevated levels of DNA oxidative damage, and selenium, zinc, and nickel are significant exposure factors.

Effects of PM2.5 and high-fat diet interaction on blood glucose metabolism in adolescent male Wistar rats: A serum metabolomics analysis based on ultra-high performance liquid chromatography/mass spectrometry

Fine particulate matter (PM2.5) and high-fat diet (HFD) are known to contribute to blood glucose metabolic disorders. However, limited research has investigated the combined impact of PM2.5 and HFD on blood glucose metabolism. This study aimed to explore the joint effects of PM2.5 and HFD on blood glucose metabolism in rats using serum metabolomics and to identify involved metabolites and metabolic pathways. The 32 male Wistar rats were exposed to filtered air (FA) or PM2.5 (real-world inhaled, concentrated PM2.5, 8 times the ambient level, ranging from 131.42 to 773.44 μg/m3) and fed normal diet (ND) or HFD for 8 weeks. The rats were divided into four groups (n = 8/group): ND-FA, ND-PM2.5, HFD-FA and HFD-PM2.5 groups. Blood samples were collected to determine fasting glucose (FBG), plasma insulin and glucose tolerance test and HOMA Insulin Resistance (HOMA-IR) index was calculated. Finally, the serum metabolism of rats was analyzed by ultra-high performance liquid chromatography/mass spectrometry (UHPLC-MS). Then we constructed the partial least squares discriminant analysis (PLS-DA) model to screen the differential metabolites, and performed pathway analysis to screen the main metabolic pathways. Results showed that combined effect of PM2.5 and HFD caused changes in glucose tolerance, increased FBG levels and HOMA-IR in rats and there were interactions between PM2.5 and HFD in FBG and insulin. By metabonomic analysis, the serum differential metabolites pregnenolone and progesterone, which involved in steroid hormone biosynthesis, were two different metabolites in the ND groups. In the HFD groups, the serum differential metabolites were L-tyrosine and phosphorylcholine, which involved in glycerophospholipid metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. When PM2.5 and HFD coexist, they may lead to more severe and complex effects on glucose metabolism by affecting lipid metabolism and amino acid metabolism. Therefore, reducing PM2.5 exposure and controlling dietary structure are important measures for preventing and reducing glucose metabolism disorders.

Assessment of the presence of UV filters and UV stabilizers in stranded dolphin blubber

The presence of ultraviolet filters (UVFs) and stabilizers (UVSs) was evaluated for the first time in the common bottlenose dolphin (Tursiops truncatus). UVFs and UVSs are compounds of growing concern because their effects on the environment are not completely known. UVFs and UVSs are added to personal care products (PCPs), such as cosmetics and products related to sun care and once released to the aquatic ecosystem, marine organisms can bioaccumulate these substances.This work aimed to determine the presence of 12 UVFs and UVSs in cetacean blubber samples to assess the pollution to which these animals of the highest trophic chain levels are exposed due to human activity. Analytical determinations were carried out using a method based on microwave-assisted extraction combined with ultrahigh-performance liquid chromatography and tandem mass spectrometry detection. The developed method was successfully applied to determine the target compounds in the blubber tissues of five necropsied common bottlenose dolphins.Three of the 12 studied compounds, namely 2-ethylhexyl 2-cyano-3,3-diphenylprop-2-enoate (octocrilene, OC), 2-hydroxy-4-methoxybenzophenone (benzophenone 3, BP3) and 3-methylbutyl (E)-3-(4methoxyphenyl) prop-2-enoate (IMC), were detected in several samples. Of the identified compounds, OC was present in all the samples and at the highest concentration within the range from 52.61 ± 18.59 to 108.0 ± 11.32 ng·g−1.

Metabolomics investigation of global responses of Cronobacter sakazakii against common sanitizing in infant formula processing environments

Cronobacter sakazakii, an opportunistic bacterium, has raised a serious outbreak in powdered infant formula recent years. In this work, four sanitizing strategies used during infant formula processing, including chlorine, quaternary ammonium chloride (QAC), 60 °C heating, and malic acid (MA), were utilized against C. sakazakii among planktonic, air-dried (A), and air-dried & washed (AW) state, followed by an exploration of the metabolic responses induced by these treatments via a dual-platform metabolomics analysis with the ultra-high performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. In the planktonic state, MA was the most effective in inhibiting bacterial growth, followed by chlorine, QAC, and 60 °C heating. Under A state, the efficacy of heating improved considerably, compared to that in the planktonic state, and remained unaltered under AW state. Chlorine and QAC were ineffective to control bacterial growth under A state, but their efficacy rose under AW state. Furthermore, the metabolomic analysis revealed chlorine induces amino acids catabolism, membrane lysis, and depression in carbohydrate and nucleotide metabolism in both planktonic and AW states, while the initiation of antioxidation mechanism was only found under AW state. Although the metabolic change caused by QAC in the planktonic state was similar to chlorine, the accumulation of osmoprotectant and membrane phospholipids within the AW cells reflected the effort to restore intracellular homeostasis upon QAC. Heating was characterized by considerable amino acid anabolism, along with mildly perturbed carbohydrate and nucleotide metabolism for heat shock protein preparation in both states. Lastly, MA promoted amino acid-dependent acid resistance under the planktonic state, and the regulation of antioxidation and osmoprotection under AW state. The metabolomics study elucidated the intracellular perturbation induced by common sanitizing, as well as the bacterial response, which provides insights for novel sanitization development.

Pharmacokinetic evaluation of Sinisan containing vinegar-processed products in depressive rats, a comprehensive perspective of ‘individual herb, herb-pair, and herbal formula’

Ethnopharmacological relevanceAs a classical formula for the treatment of depression, the clinical application of vinegar-processed products of Bupleuri Radix (Bupleurum chinense DC., BR) and Paeoniae Radix Alba (Paeonia lactiflora Pall., PRA) contained in Sinisan (SNS) is still controversial. Aim of the studyThree levels of ‘individual herb, herb-pair, and herbal formula’ were employed to investigate whether and how the processing of main drugs affected the active constituents of pharmacokinetics in SNS, as well as their impacts on the hepatic CYP450 enzyme. Materials and methodsRats were subjected to construct a chronic unpredictable mild stimulation (CUMS) model. A rapid and sensitive ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analytical method was developed and validated for simultaneously quantitative evaluation of thirteen potential active compounds of SNS in depressive rat plasma, and successfully applied to a holistic comparison of pharmacokinetics. The differences in pharmacokinetic parameters based on three different forms of drug composition from BR and PRA before and after vinegar-processing were compared. Meanwhile, qRT-PCR and Western Blot were utilized to explore the metabolic activity of three isoforms of CYP450 enzyme scattered in the livers of depressive rats. ResultsThe characteristic pharmacokinetics profiles of thirteen representative constituents in CUMS rats were influenced by vinegar-processing of BR and PRA and/or the compatibility. In detail, there were significant differences in the Cmax, AUC0-24, AUC0-∞, t1/2, and MRT0-24 of most constituents among the three different forms of drug composition from BR and PRA before and after vinegar-processing, with the most obvious changes in six constituents from the adjuvant and mediating guide drugs. And also, the pharmacokinetic parameters of seven constituents from BR and PRA in SNS containing vinegar-processed products obviously changed after compatibility. Additionally, the mRNA and protein levels of CYP1A2, CYP2E1, and CYP3A1 were observed to increase significantly with the processing of BR and PRA and the combination/formulation. ConclusionsIn conclusion, SNS containing vinegar-processed products was more conducive to the absorption of most activated constituents compared to the original formula in vivo. The vinegar-processing of BR and PRA and the compatibility co-contribute to the pharmacokinetic variability of active compounds of SNS in CUMS rats, and the extent of contribution varies among drugs, which might be related to the regulation of the hepatic drug metabolizing enzymes. The finding of the investigation could help to better understand how active compounds metabolized in vivo, which might be helpful for guiding the clinical application of SNS containing vinegar-processed products.

Optimization of ultrasonic-assisted extraction of flavonoids from Lactuca indica L. cv. Mengzao and their antioxidant properties.

In this study, the ultrasound-assisted extraction (UAE) conditions of flavonoids from Lactuca indica L.cv. Mengzao (LIM) leaves was optimized, and the flavonoids content and their antioxidant potential in different parts were analyzed. The optimal extraction parameters to obtain the highest total flavonoids content (TFC) were a a ratio of liquid to solid of 24.76 mL/g, ultrasonic power of 411.43 W, ethanol concentration of 58.86% and an extraction time of 30 min, the average TFC of LIM leaves could reach 48.01 mg/g. For the yield of flavonoids, the UAE method had the best extraction capacity compared with solvent extraction and microwave-assisted extraction (MAE). In general, the TFC in different parts of LIM followed the order flower > leaf > stem > root, the flowering period is the most suitable harvesting period. From ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) quantification, the flower samples showed significantly higher six flavonoids and had the highest radical scavenging capacities compared to other samples. A high positive correlation was observed between the antioxidant activity and TFC, luteolin-7-O-glucoside and rutin were significantly (p < 0.05) correlated with all antioxidant evaluations. This study provides valuable information for the development and utilization of flavonoids in Lactuca indica as ingredients in food, feed and nutritional health products.

Preparative Fractionation of Phenolic Compounds and Isolation of an Enriched Flavonol Fraction from Winemaking Industry By-Products by High-Performance Counter-Current Chromatography.

High-performance counter-current chromatography (HPCCC) was used as a tool for the isolation and fractionation of phenolic compounds (PCs) in extracts from wine lees (WL) and grape pomace (GP). The biphasic solvent systems applied for HPCCC separation were n-butanol:methyl tert-butyl ether:acetonitrile:water (3:1:1:5) with 0.1% trifluoroacetic acid (TFA) and n-hexane:ethyl acetate:methanol:water (1:5:1:5). After refining the ethanol:water extracts of GP and WL by-products by ethyl acetate extraction, the latter system yielded an enriched fraction of the minor family of flavonols. Recoveries of 112.9 and 105.9 mg of purified flavonols (myricetin, quercetin, isorhamnetin, and kaempferol) in GP and WL, respectively, from 500 mg of ethyl acetate extract (equivalent to 10 g of by-product) were obtained. The HPCCC fractionation and concentration capabilities were also exploited for the characterization and tentative identification of constitutive PCs by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). In addition to the isolation of the enriched flavonol fraction, a total of 57 PCs in both matrixes were identified, 12 of which were reported for the first time in WL and/or GP. The application of HPCCC to GP and WL extracts may be a powerful approach to isolate large amounts of minor PCs. The composition of the isolated fraction demonstrated quantitative differences in the individual compound composition of GP and WL, supporting the potential exploitation of these matrixes as sources of specific flavonols for technological applications.

Simultaneous quantitation of ketamine, norketamine and dehydronorketamine in human milk using a novel ultra high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) assay

There is a paucity of data on the transfer of ketamine from maternal blood into human milk. Quantification of ketamine in human milk provides information about the potential exposure of the infant to ketamine and its metabolites from the mother during lactation. A highly specific, reproducible, and sensitive UPLC-MS/MS based analytical method was developed and validated for the quantitation of ketamine and its metabolites (norketamine and dehydronorketamine) in human milk. Samples were subjected to a simple protein precipitation and ketamine-d4 and norketamine-d4 were used as internal standards. Separation of the analytes was achieved using an Acquity UPLC equipped with BEH RP18 1.7 µm, 2.1 × 100 mm column. Mass spectrometric analysis of the analyte ions was carried out using electrospray with positive ionization and multiple reaction monitoring mode. The assay was linear over a concentration range of 1–100 ng/mL for ketamine and norketamine, and 0.1–10 ng/mL for dehydronorketamine. Acceptable intra-day and inter-day accuracy and precision were observed for all the analytes. High recovery of the analytes and minimal matrix effect were observed. Stability of analytes was confirmed at the tested conditions. This assay was successfully used to measure analytes in human milk samples collected from lactating women enrolled in a clinical research study. This is the first validated method that simultaneously quantified ketamine and its metabolites in human milk.

Profiling of branched chain and straight chain saturated fatty acids by ultra-high performance liquid chromatography-mass spectrometry

Branched chain fatty acids (BCFAs) are one of the important sub categories of fatty acids (FAs) which have unique functions in nature. They are commonly analyzed by GC–MS after derivatization to methyl esters (FAMEs). On the other hand, there is a lack of isomer-selective LC-MS methods which allow the distinction of different isomers with wide coverage of carbon chain length. In this work, a systematic retention and isomer selectivity study on seven commercially available UHPLC columns (six polysaccharide columns Chiralpak IA-U, IB-U, IC-U, ID-U, IG-U and IH-U; one Acquity UPLC CSH C18 column) was performed. Various experimental factors were evaluated including column temperatures, gradient profiles and flow rates to elucidate their effects on the separation ability of homologous series of BCFAs with distinct chain lengths, different branching types and branching positions. In general, IG-U outperformed the other columns in terms of isomer selectivity especially for the short and medium-chain BCFA isomers while RP C18 showed good potential in terms of selectivity for long-chain BCFA isomers. Furthermore, after the evaluation of the chromatographic retention pattern on the various columns and method optimization, we report a methodology for untargeted isomer-selective BCFA profiling without precolumn derivatization with UHPLC-ESI-MS/MS by quadrupole-time-of-flight instrument with SWATH acquisition. The best method provides selectivity for constitutional isomers of BCFAs covering distinct chain length (C5-C20) with different branching types (methyl or ethyl) and branching positions (2Me, 3Me, 4Me, 6Me, anteiso and iso-BCFAs) with an optimized LC condition on Acquity UPLC CSH C18 column. Finally, the optimized method was applied for the BCFAs profiling in lipid extracts of Staphylococcus aureus samples. Besides, pooled human platelets and pooled human plasma were evaluated as mammalian samples for presence of BCFAs as well. The new method showed strong potential for BCFA profiling in bacterial samples including different isomers anteiso and iso-BCFAs, which could be a useful tool for related subdisciplines in metabolomics and lipidomics in particular in combination with electron-activated dissociation MS. Compared to GC, the presented isomer selective LC methods can be also of great utility for preparative purposes. Equivalent (carbon) chain length numbers were calculated for RP18 and Chiralpak IG-U and compared to those of FAMEs obtained by GC.

Comparative MS- and NMR-Based Metabolome Mapping of Egyptian Red and White Squill Bulbs F. Liliaceae and in Relation to Their Cytotoxic Effect.

Urginea maritima L. (squill) species is widely spread at the Mediterranean region as two main varieties, i.e., white squill (WS) and red squill (RS), that are recognized for several health potentials. The major secondary metabolite classes of the squill are cardiac glycosides, mainly, bufadienolides, flavonoids, and anthocyanins. Herein, a multiplex MS and NMR metabolomics approach targeting secondary and aroma compounds in WS and RS was employed for varieties classification. Solid-phase micro extraction-gas chromatography/mass spectroscopy (SPME-GC/MS), ultra-high-performance liquid chromatography/mass spectrometry (UPLC/MS), as well as nuclear magnetic resonance (NMR) provided fingerprinting and structural confirmation of the major metabolites for both types of the squill. For comparison of the different platforms' classification potential, multivariate data analysis was employed. While Bufadienolides, viz. "hydroxy-scilliglaucosidin-O-rhamnoside, desacetylscillirosidin-O-rhamnoside and bufotalidin-O-hexoside" as well as oxylipids, were enriched in WS, flavonoids, i.e., dihydro-kaempferol-O-hexoside and its aglycon, taxifolin derivative, were predominant in RS. A cytotoxicity screening against three cancer cell lines, including breast adenocarcinoma (MCF-7), lung (A-549), and ovarian (SKOV-3) cell lines was conducted. Results revealed that WS was more effective on A-549 and SKOV-3 cell lines (WS IC50 0.11 and 0.4 µg/mL, respectively) owing to its abundance of bufadienolides, while RS recorded IC50 (MCF7 cell line) 0.17 µg/mL since is is rich inflavonoids.

Comprehensive Analysis of Microbiome, Metabolome, and Transcriptome Revealed the Mechanisms of Intestinal Injury in Rainbow Trout under Heat Stress.

Global warming is one of the most common environmental challenges faced by cold-water fish farming. Intestinal barrier function, gut microbiota, and gut microbial metabolites are significantly altered under heat stress, posing serious obstacles to the healthy artificial culture of rainbow trout. However, the molecular mechanisms underlying intestinal injury in rainbow trout under heat stress remain unclear. In the present study, the optimal growth temperature for rainbow trout (16 °C) was used for the control group, and the maximum temperature tolerated by rainbow trout (24 °C) was used for the heat stress group, which was subjected to heat stress for 21 days. The mechanism of intestinal injury in rainbow trout under heat stress was explored by combining animal histology, 16S rRNA gene amplicon sequencing, ultra-high performance liquid chromatography-mass spectrometry, and transcriptome sequencing. The results showed that the antioxidant capacity of rainbow trout was enhanced under heat stress, the levels of stress-related hormones were significantly increased, and the relative expression of genes related to heat stress proteins was significantly increased, indicating that the heat stress model of rainbow trout was successfully established. Secondly, the intestinal tract of rainbow trout showed inflammatory pathological characteristics under heat stress, with increased permeability, activation of the inflammatory factor signaling pathway, and increased relative expression of inflammatory factor genes, suggesting that the intestinal barrier function was impaired. Thirdly, heat stress caused an imbalance of intestinal commensal microbiota and changes in intestinal metabolites in rainbow trout, which participated in the stress response mainly by affecting lipid metabolism and amino acid metabolism. Finally, heat stress promoted intestinal injury in rainbow trout by activating the peroxisome proliferator-activated receptor-α signaling pathway. These results not only expand the understanding of fish stress physiology and regulation mechanisms, but also provide a scientific basis for healthy artificial culture and the reduction of rainbow trout production costs.

Pollution Characteristics and Risk Assessment of Typical Endocrine Disrupting Chemicals in Drains Flowing into the Yellow River of Ningxia

Solid-phase extraction and ultra-high performance liquid chromatography-mass spectrometry were used to detect and analyze the contamination of nine typical endocrine disrupting chemicals (EDCs) in drains flowing into the Yellow River of Ningxia. Sources of these EDCs were analyzed, and their risks were assessed. The results showed that EDCs were detected in drains at all 33 sampling sites, with total concentrations （ΣEDCs） of 82.28-1730.09 ng·L-1. Among phenolic compounds, bisphenol A (BPA) and nonylphenol (NP) were two that were more commonly detected EDCs, with the detection rates above 90%; estrone (E1) and estriol (E3) were more commonly detected estrogenic compounds, both with detection rates of 79%. On a spatial scale, the average concentrations of ΣEDCs in the drains in Shizuishan and Yinchuan were much higher than those in Wuzhong and Zhongwei. Concentrations of ΣEDCs at the sampling sites before flowing into Yellow River ranged from 82.28 to 979.82 ng·L-1. The source analysis showed that industrial wastewater and domestic sewage were two primary sources for BPA, whereas industrial wastewater was the primary source for OP. The primary sources of E1 and E3 were livestock and poultry breeding wastewater and domestic sewage, respectively. Risk assessment results showed that EDCs in drains flowing into the Yellow River posed low or moderate ecological risk but high risk for estrogenic activity at all sampling sites.

Untargeted UHPLC-MS Metabolomics Reveals the Metabolic Perturbations of Helicoverpa armigera under the Stress of Novel Insect Growth Regulator ZQ-8

According to the previous research of our group, we found compound ZQ-8 ((1S,2R,4S)-1,3,3-trimethylbicyclo [2.2.1]heptan-2-yl-4-(tert-butyl)benzoate). This compound showed a strong growth inhibitory effect on Helicoverpa armigera by inhibiting chitinase 2 and endochitinase. To further understand the mechanism of ZQ-8 interfering with the growth and development of H. armigera, ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was utilized to analyze the metabolomics of the epidermis and viscera of H. armigera after ZQ-8 stress. The results revealed that the content of most metabolites was down-regulated after ZQ-8 treatment. Through the analysis of metabolic pathways, it was found that ZQ -8 mainly interfered with energy metabolism and amino acid biosynthesis pathways, which may be one of the important factors in which ZQ-8 caused the death of H. armigera larvae. Furthermore, ZQ-8 not only inhibits chitin degradation but also inhibits chitin synthesis in vivo. These findings provide new insights into a better understanding of the mechanism of action of ZQ-8.

Cytotoxic Effects and Oxidative Stress Produced by a Cyanobacterial Cylindrospermopsin Producer Extract versus a Cylindrospermopsin Non-Producing Extract on the Neuroblastoma SH-SY5Y Cell Line.

The incidence and interest of cyanobacteria are increasing nowadays because they are able to produce some toxic secondary metabolites known as cyanotoxins. Among them, the presence of cylindrospermopsin (CYN) is especially relevant, as it seems to cause damage at different levels in the organisms: the nervous system being the one most recently reported. Usually, the effects of the cyanotoxins are studied, but not those exerted by cyanobacterial biomass. The aim of the present study was to assess the cytotoxicity and oxidative stress generation of one cyanobacterial extract of R. raciborskii non-containing CYN (CYN-), and compare its effects with those exerted by a cyanobacterial extract of C. ovalisporum containing CYN (CYN+) in the human neuroblastoma SH-SY5Y cell line. Moreover, the analytical characterization of potential cyanotoxins and their metabolites that are present in both extracts of these cultures was also carried out using Ultrahigh Performance Liquid Chromatography-Mass Spectrometry, in tandem (UHPLC-MS/MS). The results show a reduction of cell viability concentration- and time-dependently after 24 and 48 h of exposure with CYN+ being five times more toxic than CYN-. Furthermore, the reactive oxygen species (ROS) increased with time (0-24 h) and CYN concentration (0-1.11 µg/mL). However, this rise was only obtained after the highest concentrations and times of exposure to CYN-, while this extract also caused a decrease in reduced glutathione (GSH) levels, which might be an indication of the compensation of the oxidative stress response. This study is the first one performed in vitro comparing the effects of CYN+ and CYN-, which highlights the importance of studying toxic features in their natural scenario.