Ultra-high Performance Liquid Chromatography Coupled To High-resolution Mass Spectrometry Research Articles

Industrial upcycling of almond skin through production of novel brioches

The global sustainability policy emphasizes reusing of agri-food waste and by-products to enhance food bioactive properties. Thus, brioches were processed incorporating almond skin powder (ASP): control (CTR), without ASP addition; 5-ASP, with 5% (w/w) ASP; and 10-ASP, with 10% (w/w) ASP. Seven different brioches shapes were obtained for each recipe. Flavonoids were mainly detected in Tuono almond skin by Ultra-High Performance Liquid Chromatography coupled to High-Resolution Mass Spectrometry (UHPLC-HRMSMS), in particular, flavan-3-ol monomers. The ethanolic extract of Tuono almond skins contained polar lipids (oxylipins and phospholipids). Gas Chromatography–Mass Spectrometry (GC-MS) identified six major fatty acids, mainly oleic acid (48.01%). Photothermal degradation impact on bioactive compounds was evaluated using a first-order kinetic model. Antioxidant activity was studied using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,2-diphenyl-1-picrylhydrazyl and β-carotene bleaching test. α-amylase, α-glucosidase, and lipase inhibitory effect were also tested. The acidification of the doughs was consistent across all trials. Lactic acid bacteria and yeast levels increased. Importantly, the final products were free from undesirable microorganisms. The addition of ASP led to reduced weight loss and specific volume for all seven brioche types. Furthermore, the firmness, crumb structure, and sensory profile of the final products were noticeably influenced. Tasters clearly favoured the Treccina brioches. The production of sweet leavened baked goods was carried out in triplicate in two independent experiments. The statistical model applied to the data considered the effects of brioche shape and the addition of ASP. Kinetic data revealed that the half-life extension for both total phenol and flavonoid content was observed in the 10-ASP sample (18.00382). 10-ASP sample exhibited promising ABTS radical scavenging activity, with inhibitory concentration 50% (IC50) values of 18.64 mg/mL after 9 days of photothermal degradation. Moreover, when testing 10-ASP Treccina against α-amylase and α-glucosidase, the IC50 values were 198.16 and 190.23 μg/mL, respectively, even after 9 days.

Tracing the biographies of textiles in the transition of medieval to modern times: Wool fabrics and brigandines from an Iberian castle

Findings of archaeological textiles and fibres in Northern Iberia are extremely rare. The occurrence of a set of textile fragments, dated between the 14th and 16th centuries CE at the Pambre castle (Palas de Rei, Lugo, Spain) is exceptional. The original stone roof of the southeastern tower was intact. The dark, cold and moist conditions inside the tower favoured the preservation of a unique series of waterlogged textile remains. In addition, a set of pseudomorphs preserved by mineral replacement were recovered from the east edge of the north wing in the main hall of the castle. Fibres have been identified using optical and scanning electron microscopy (SEM), and they have been chemically characterised using Energy Dispersive X-Ray Spectroscopy (EDX). We also performed analytical pyrolysis-GC-MS (Py-GC-MS) and thermally assisted hydrolysis and methylation (THM-GC-MS) of the wool fabrics and pseudomorphs to assess their state of degradation and the presence of chemical markers associated to the use of these textile remains. High performance liquid chromatography with diode array detection (HPLC-DAD-MS) and ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) analysis were applied on wool fabrics to identify the chemical markers of dyes but without success. To expand the information related to raw material identification and the technical aspects of the fabrics, further evidence such as adherences identified as opal phytoliths, seeds, and insect remains associated to wool fabrics were examined. These findings offer a unique glimpse into the clothing dated to the end of the Medieval period, and its life-cycle. Wool scraps were probably part of at least two different garments, whereas the mineralised textiles probably formed part of at least two brigandines which were made of bast fibres, flax, or hemp.

Evaluation of the Antioxidant, Photoprotective and Wound Healing Capacity of Guazuma ulmifolia lam. Extracts in L-929 Cells Exposed to UV-A and UV-B Irradiation

The UV irradiation causes the generation of reactive oxygen species in the skin that mediate oxidative damage, favoring photoaging and skin cancer. Photoaging can be mitigated with the use of plants with antioxidant capacity. This study sought to evaluate the photoprotection capacity of two extracts of Guazuma ulmifolia Lam., aqueous (G1) and acetonic (G2), on UV-A and UV-B irradiated L-929 cells, besides the wound healing capacity. In the antioxidant evaluation, it was found that the extracts have great antioxidant potential. In the irradiation tests, G2 increased cell viability by 19.99%. Furthermore, both extracts have shown wound healing capacity. There was a significant restoration of the superoxide dismutase and catalase enzymatic activity by G2 and through ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS), two antioxidant compounds were identified: (epi)catechin and procyanidin. The results showed that the extracts are promising antioxidants, and may collaborate in wound healing, with emphasis to G2 which protected cells from UV-A irradiation.

Effects of Different Heating Treatments on the Antioxidant Activity and Phenolic Compounds of Ecuadorian Red Dacca Banana.

The banana is a tropical fruit characterized by its composition of healthy and nutritional compounds. This fruit is part of traditional Ecuadorian gastronomy, being consumed in a wide variety of ways. In this context, unripe Red Dacca banana samples and those submitted to different traditional Ecuadorian heating treatments (boiling, roasting, and baking) were evaluated to profile their phenolic content by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) and the antioxidant activity by ORAC, ABTS, and DPPH assays. A total of sixty-eight phenolic compounds were identified or tentatively identified in raw banana and treated samples, highlighting the content in flavonoids (flavan-3-ols with 88.33% and flavonols with 3.24%) followed by the hydroxybenzoic acid family (5.44%) in raw banana samples. The total phenolic compound content significantly decreased for all the elaborations evaluated, specifically from 442.12 mg/100 g DW in fresh bananas to 338.60 mg/100 g DW in boiled (23.41%), 243.63 mg/100 g DW in roasted (44.90%), and 109.85 mg/100 g DW in baked samples (75.15%). Flavan-3-ols and flavonols were the phenolic groups most affected by the heating treatments, while flavanones and hydroxybenzoic acids showed higher stability against the heating treatments, especially the boiled and roasted samples. In general, the decrease in phenolic compounds corresponded with a decline in antioxidant activity, evaluated by different methods, especially in baked samples. The results obtained from PCA studies confirmed that the impact of heating on the composition of some phenolic compounds was different depending on the technique used. In general, the heating processes applied to the banana samples induced phytochemical modifications. Even so, they remain an important source of bioactive compounds for consumers.

Ultra-high performance liquid chromatography high-resolution mass spectrometry for metabolomic analysis of dental calculus from Duke Alessandro Farnese and Maria D’Aviz

Dental calculus is a valuable resource for the reconstruction of dietary habits and oral microbiome of past populations. In 2020 the remains of Duke Alessandro Farnese and his wife Maria D’Aviz were exhumed to get novel insights into the causes of death. This study aimed to investigate the dental calculus metabolome of the noble couple by untargeted metabolomics. The pulverized samples were decalcified in a water-formic acid mixture, extracted using methanol/acetonitrile and analyzed by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) using a reversed-phase separation followed by electrospray ionization and full scan in positive and negative ion mode. Waters Synapt-G2-Si High-Definition hybrid quadrupole time-of-flight mass spectrometer was used. Significant features were then identified using MSE acquisition mode, recording information on exact mass precursor and fragment ions within the same run. This approach, together with data pre-treatment and multivariate statistical analysis allowed for the identification of compounds able to differentiate between the investigated samples. More than 200 metabolites were identified, being fatty acids, alcohols, aldehydes, phosphatidylcholines, phosphatidylglycerols, ceramides and phosphatidylserines the most abundant classes. Metabolites deriving from food, bacteria and fungi were also determined, providing information on the habits and oral health status of the couple.

Evaluation of Pharmacokinetic and Toxicological Parameters of Arnica Tincture after Dermal Application In Vivo.

Cutaneous leishmaniasis (CL) is classified as a neglected tropical disease by the World Health Organization. As the standard drugs for the treatment of this disease suffer from severe unwanted effects, new effective and safe therapeutic options are required. In our previous work, Arnica tincture showed promising antileishmanial effects in vitro and in vivo. For the potential treatment of human CL patients with Arnica tincture, data on the pharmacokinetic properties of the bioactive, antileishmanial compounds (the sesquiterpene lactone (STL) helenalin and its derivatives) are needed. Therefore, we studied the in vivo absorption of the bioactive compounds after the dermal application of Arnica tincture in rats. Moreover, we analyzed the blood plasma, urine, and feces of the animals by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Although the majority (84%) of the applied STLs (1.0 mg) were absorbed, the concentrations in the plasma, urine, and feces were below the limit of detection (0.3 ng/mL) in the samples for UHPLC-HRMS analysis. This result may be explained by extensive metabolism and slow permeation accompanied by the accumulation of STLs in the skin, as described in our previous work. Accordingly, the plasma concentration of STLs after the topical application of Arnica tincture was very far from a dose where toxicity could be expected. Additionally, tests for corrosive or irritant activity as well as acute and repeated-dose dermal toxicity did not show any positive results after the administration of the amounts of Arnica tincture that would be needed for the treatment of CL. Consequently, in the treatment of CL patients with Arnica tincture, no toxic effects are expected, other than the known sensitization potential of the STLs.

Targeted screening and quantification of synthetic cathinones and metabolites in hair by UHPLC-HRMS.

Synthetic cathinones (SCs) are new psychoactive substances with sympathomimetic effects, which emerged into the illegal drug market to replace controlled stimulants. Since every year more powerful and toxic substances enter the illicit market, there is the need for analytical methodologies able to detect these new compounds in conventional and non-conventional biological matrices. We sought to develop and validate a targeted screening and quantification method for thirty-two parent SCs and two metabolites in hair samples by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). 20 mg hair samples were soaked in 250 µL of 2 mM ammonium formate, methanol and acetonitrile mixture (50/25/25, v/v/v) and incubated overnight at 40°C. After incubation, the samples were evaporated to dryness under nitrogen stream and reconstituted with 100 µL of mobile phase mix (A:B, 80:20) and 10 µL were injected into UHPLC-HRMS. A Q ExactiveTM Focus Orbitrap Mass spectrometer with full scan and targeted data-dependent MS/MS scan acquisition was used for the screening and quantitation analysis. The assay was linear from 5 to 500 pg/mg hair for all the analytes under investigation. Intra-day and inter-day precision were always < 15% and matrix effect and analytical recovery were always within acceptable criteria (±25% and >50%, respectively). The developed method was applied to authentic hair samples from SCs consumers. The most prevalent found SCs were 3,4-Methylenedioxy-α-Pyrrolidinohexanophenone with a concentration range of 6.0-1,000.0 pg/mg along with α-Pyrrolidinohexiophenone (54.0 and 554.0 pg/mg, respectively), 3-Methylmetcathinone (556.0 and 5,000.0 pg/mg) and 4-Methylethcathinone (11.5 and 448.0 pg/mg) CONCLUSIONS: The developed method showed good selectivity, specificity, an easy and low-cost sample preparation and an analysis time compatible with a high throughput laboratory.

Preliminary authentication of apple juices using untargeted UHPLC-HRMS analysis combined to chemometrics

In this work, apple juice samples from different farming and production processes (direct and concentrated juices; organic and conventional juices) were analyzed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). A workflow was developed and implemented for data processing using the Workflow4Metabolomics (W4M) platform. First, features were detected using XCMS, and next data filtration steps were applied leading to the removal of nearly 50% of the detected features. Intra- and inter-batch correction was then performed, followed by chemometric tools (PCA, PLS-DA, OPLS-DA, ANOVA). The developed approach successfully discriminated apple juice samples in two distinct scenarios simultaneously (direct vs. concentrated juices and organic vs. conventional juices). PCA highlighted the reproducibility of the method and confirmed the efficiency of batch corrections. OPLS-DA models showed good quality metrics, particularly after feature selection for organic vs. conventional juices discrimination (almost 80% of predictive ability). Based on ANOVA and OPLS-DA results, 24 features were retained as significantly discriminant. Among them, some compounds were identified as amino-acids and derivatives, using additional MS/MS experiments and online databases. An independent data set was used to evaluate their potential as marker compounds, with promising results obtained. Further investigation is needed to validate such an untargeted method and its routine application to detect apple juice adulteration and confirm its authenticity.

Lipidomic profiling of Skipjack tuna (Katsuwonus pelamis) by ultrahigh-performance liquid chromatography coupled to high resolution mass spectrometry

A method of ultrahigh performance liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS) was established for characterization of the lipid profile of Skipjack tuna. Over 300 lipid molecular species were identified through cross-acquisition in both positive and negative ion mode. Phospholipids (PLs) were dominant in Skipjack tuna. Lysophosphatidylethanolamine (LPE), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) were the main lipid molecular species in PLs, accounting for 89.24% of the total PLs. The ratio of sphingolipids (SLs) and glycerolipids (GLs) were considerable, accounting for 12.30% and 13.60% of the total lipids respectively. Ceramide (Cer) was the main lipid molecular species of SLs, accounting for 64.96% of total SLs, followed by sphingomyelin (SM), accounting for 25.45% of total SLs. Ether diglycerides (ether DG) were the main lipid molecular species of GLs (97.83%). The main fatty acids (FAs) are unsaturated fatty acids (UFAs) in Skipjack tuna. Besides, a new FAs class branched fatty acid esters of hydroxy fatty acids (FAHFA) was detected, together with the FA. The active lipids identified in this study can be used to evaluate the nutritional value of Skipjack tuna.

Suspected-screening assessment of the occurrence of organic compounds in sewage sludge

The profiling of emerging organic pollutants present in sludge and generated during wastewater treatment is much more limited than in water. This is mainly due to the difficulty of sludge analysis because of its high content of organic matter and interfering compounds. In this study, a generic extraction method using a mixture of buffered water (pH 4.1) and solid phase extraction (SPE) clean-up was applied to samples of sludge obtained in different treatment plants. This extraction was followed by determination of the contaminants by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS), using suspected screening to detect the most relevant organic compounds that access the environment through sludge application. This screening (including >3000 substances, such as, pharmaceuticals, pesticides, metabolites and industrial chemicals) tentatively identified 122 compound and assigned most probable structure to 39. The set of compounds assigned to a probable structure was increased in 14 compounds by searching in a free database of metabolites. Fifteen compounds were unequivocally confirmed against the analytical standard. Pharmaceuticals and personal care products (PPCPs), with 31 substances identified and 8 confirmed were the main group of compounds. Compounds frequently detected in all sludge samples include nucleotides such as adenosine triphosphate, amino acids such as phenylalanine, or peptides such as leu-phe. Altogether, the results of this work highlight the interest of HRMS to draw the profile of organic compounds in complex matrices.

Detailed investigation of the composition and transformations of phenolic compounds in fresh and fermented Vaccinium floribundum berry extracts by high-resolution mass spectrometry and bioinformatics.

IntroductionBlueberries are known for their very high content of biologically active phenolic compounds; nonetheless, differently from the North American and European species of blueberries, Neotropical blueberries have not been extensively studied yet.ObjectivesIn the present paper, the phenolic composition of Vaccinium floribundum Kunth, which is endemic to the Andean regions and grows 1,600 to 4,500 meters above sea level, was investigated by ultra‐high‐performance liquid chromatography coupled to high‐resolution mass spectrometry (UHPLC‐HRMS). Native and fermented berries were compared in terms of phenolic composition as well as antioxidant activity, total phenolic content, and total anthocyanin content.Materials and Methods V. floribundum native and fermented berries were extracted and analyzed by UHPLC‐HRMS. The acquired datasets were processed by Compound Discoverer 3.1 using a dedicated data analysis workflow that was specifically set up for phenolic compound identification.ResultsIn total, 309 compounds were tentatively identified, including anthocyanins, flavonoids, phenolic acids, and proanthocyanidins. The molecular transformations of phenolic compounds during fermentation were comprehensively investigated for the first time, and by a customized data processing workflow, 13 quinones and quinone methides were tentatively identified in the fermented samples. Compared to other species of the genus Vaccinium, a peculiar phenolic profile is observed, with low abundance of highly methylated compounds.ConclusionAndean berries are a rich source of a wide variety of phenolic compounds. Untargeted MS analyses coupled to a dedicated data processing workflow allowed expanding the current knowledge on these berries, improving our understanding of the fate of phenolic compounds after fermentation.

Site-specific protein modification by 3-n-butylphthalide in primary hepatocytes: Covalent protein adducts diminished by glutathione and N-acetylcysteine

Aims3-n-Butylphthalide (NBP) is widely used for the treatment of cerebral ischaemic stroke but can causeliver injury in clinical practice. This study aims to elucidate the underlying mechanisms and propose potential preventive strategies. Main methodsNBP and its four major metabolites, 3-hydroxy-NBP (3-OH-NBP), 10-hydroxy-NBP, 10-keto-NBP and NBP-11-oic acid, were synthesized and evaluated in primary human or rat hepatocytes (PHHs, PRHs). NBP-related substances or amino acid adducts were identified and semi-quantitated by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The target proteins and binding sites were identified by shotgun proteomics based on peptide mass fingerprinting coupled with tandem mass spectrometry and verified by molecular docking. Key findingsThe toxicity of NBP and its four major metabolites were compared in both PHHs and PRHs, and 3-OH-NBP was found to be the most toxic metabolite. 3-OH-NBP induced remarkable cell death and oxidative stresses in hepatocytes, which correlated well with the levels of glutathione and N-acetylcysteine adducts (3-GSH-NBP and 3-NAC-NBP) in cell supernatants. Additionally, 3-OH-NBP covalently conjugated with intracellular Cys, Lys and Ser, with preferable binding to Cys sites at Myh9 Cys1380, Prdx4 Cys53, Vdac2 Cys48 and Vdac3 Cys36. Furthermore, we found that CYP3A4 induction by rifampicin augmented NBP-induced cell toxicity and supplementing with GSH or NAC alleviated the oxidative stresses and reactive metabolites caused by 3-OH-NBP. SignificanceOur work suggests that glutathione depletion, mitochondrial injury and covalent protein modification are the main causes of NBP-induced hepatotoxicity, which may be prevented by exogenous GSH or NAC supplementation and avoiding concomitant use of CYP3A4 inducers.

Application of an innovative metabolomics approach to discriminate geographical origin and processing of black pepper by untargeted UHPLC-Q-Orbitrap-HRMS analysis and mid-level data fusion

An untargeted metabolomics approach based on ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) fingerprinting was applied to investigate the metabolic differences of black pepper among three geographical origins (Sri Lanka, Vietnam, and Brazil) and two post-harvest processing (sterilized and non-sterilized spice). Principal component analysis (PCA) was employed to assess the overall clustering of samples, whereas supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was effectively used for discrimination purposes. OPLS-DA models were fully validated (R2Y and Q2 values > 0.5) and the variable importance in projection (VIP) approach was employed to provide valuable data about differential metabolites with high discrimination potential (8 markers were putatively identified). For origin differentiation, three markers were highlighted with VIP values > 1.5 (i.e. reynosin, artabsinolide D, and tatridin B). Fatty acid derivates were the most frequent markers within the metabolites annotated for processing discrimination (e.g. 10,16-dihydroxyhexadecanoic acid and 9-hydroperoxy-10E-octadecenoic acid). Additionally, different combinations of mid-level data fusion of chromatographic-mass spectrometric techniques (UHPLC and gas chromatography coupled to HRMS) and proton nuclear magnetic resonance spectroscopy (1H NMR) were evaluated for the first time for geographical and processing discrimination of black pepper. The NMR-UHPLC-GC mid-level fused model was preferred among the tested fusion approaches since good sample clustering and no misclassification were achieved. Enhanced correct classification rate was achieved by mid-level data fusion compared with the findings obtained for one of the individual techniques (1H NMR fingerprinting) (from 92% to 100% of samples correctly classified). This study opens the path to new metabolomics approaches for black pepper authentication and quality control.

α-glucosidase inhibitors from Syzygium polyanthum (Wight) Walp leaves as revealed by metabolomics and in silico approaches

Ethnopharmacological relevanceSyzygium polyanthum (Wight) Walp leaves are traditionally used to cure diabetes in many regions of Indonesia. Traditional use involves boiling the leaves until the water is reduced to half volume, and then the decoction is taken 1–2 times daily. Despite several studies reporting the antidiabetic activity of this plant, bioactive compounds have not been well identified. Aim of the studyIndonesia is one of the countries with the highest diabetes cases, particularly type 2 diabetes mellitus (T2DM). Few people have access to modern medicinal treatment; thus, the role of antidiabetic traditional medicine has become increasingly important. This research aimed to identify α-glucosidase inhibitors from S. polyathum leaves using a metabolomics approach. When the active compounds of S. polyathum are properly identified, the quality of the herb can be more easily controlled. Materials and methodsThe dried leaves of S. polyanthum were extracted by a comprehensive extraction method using a solvent combination of n-hexane, acetone, and water in a gradient, resulting in a total of 42 fractions. All fractions were subjected to an in vitro α-glucosidase inhibition test and chemical profile analysis using Nuclear Magnetic Resonance (NMR) and high performance liquid chromatography (HPLC). Orthogonal projection least square (OPLS) analysis was used to correlate the two data to identify NMR signals, and HPLC chromatogram peaks correlated to the activity. 2D NMR and ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) analyses were also used to give more precise compound identification. The activity of the identified active compounds was confirmed by an in silico technique. Results and discussionThe results of the α-glucosidase activity test showed that the most active fractions were obtained from solvents with medium polarity: Fractions 9 and 10 (F9 and F10), obtained from gradient acetone-water 4:1 and 3:2, respectively. The IC50 values of F9 and F10 were 24.8 and 31.8 μg/mL, respectively. NMR data showed that F9 had more intense and diverse signals in the aromatic region than F10. OPLS analysis results showed that some typical flavonoid signals abundant in F9 positively correlated with α-glucosidase activity. 2D NMR and UHPLC-HRMS analysis of F9 led to the conclusion that these signals could be attributed to myricetin-3-O-rhamnoside (myricitrin) and epigallocatechin-3-gallate (EGCG). In silico analysis confirmed these results, as myricitrin and EGCG had binding energies resembling acarbose as a positive control (-8.47, -8.19, and -10.13, respectively). ConclusionsNMR and HPLC-metabolomics successfully identified myricitrin and EGCG as α-glucosidase inhibitors from S. polyanthum leaves, and docking analysis validated their inhibitory activity. The results of this study justified the traditional use of S. polyanthum as an antidiabetes herbal.

Thermal degradation of stigmasterol under the deodorisation temperature exposure alone and in edible corn oil

Phytosterols are commonly found in vegetable oils and possess health benefits for humans. While investigating the chemical conversion of stigmasterol at deodorisation temperatures, gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) experiments led to the identification of 5-ethyl-6-methyl-3-heptene-2-one, 3-hydoxy-steroid, 3-ketostigmasterol, and 3,7-diketostigmasterol as by-products. The identification of these compounds assisted in the interpretation of the stigmasterol oligomers characterised by high-pressure size exclusion chromatography (HPSEC). A similar analysis was conducted in stripped corn oil at the deodorisation temperatures. As such, 5-ethyl-6-methyl-3-heptene-2-one, 3-hydoxy-steroid, 3-ketostigmasterol and 3,7-diketostigmasterol were also detected in stripped corn oil, while the contents of 3-hydoxy-steroid and 5-ethyl-6-methyl-3-heptene-2-one were higher than those of 3-ketostigmasterol, as revealed by quantum chemical simulations. In addition, stripped corn oil exhibited the characteristic of preventing stigmasterol degradation below 200 °C, whereas it enhanced the chemical conversion (such as esterification and degradation) of stigmasterol at higher temperatures.

A Metabolomics Approach and Chemometric Tools for Differentiation of Barley Cultivars and Biomarker Discovery.

One of the ultimate goals of plant breeding is the development of new crop cultivars capable of withstanding increasing environmental stresses, to sustain the constantly growing population and economic demands. Investigating the chemical composition of the above and underground tissues of cultivars is crucial for the understanding of common and specific traits thereof. Using an untargeted metabolomics approach together with appropriate chemometrics tools, the differential metabolite profiles of leaf and root extracts from five cultivars of barley (‘Erica’, ‘Elim’, ‘Hessekwa’, ‘S16’ and ‘Agulhas’) were explored and potential signatory biomarkers were revealed. The study was conducted on seedlings grown for 21 days under identical controlled conditions. An ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) was employed to analyse hydromethanolic leaf and root extracts of barley cultivars. Furthermore, unsupervised and supervised learning algorithms were applied to mine the generated data and to pinpoint cultivar-specific metabolites. Among all the classes of metabolites annotated, phenolic acids and derivatives formed the largest group and also represented the most discriminatory metabolites. In roots, saponarin, an important allelochemical differentially distributed across cultivars, was the only flavonoid annotated. The application of an untargeted metabolomics approach in phenotyping grain crops such as barley was demonstrated, and the metabolites responsible for differentiating between the selected cultivars were revealed. The study provides insights into the chemical architecture of barley, an agro-economically relevant cereal crop; and reiterates the importance of metabolomics tools in plant breeding practices for crop improvement.

Pterocarpus santalinus L. extract mitigates gamma radiation-inflicted derangements in BALB/c mice by Nrf2 upregulation

Plant-based natural extracts contain several nutrients and bioactive compounds, such as phenolics and flavonoids, that possess various health-promoting activities. This study investigated the effects of polyphenols from Pterocarpus santalinus hydroalcoholic extract (PSHE) against gamma radiation-induced derangements via the upregulation of Nrf2. Ultra High Performance Liquid Chromatography Coupled to High Resolution Mass Spectrometry (UHPLC-HRMS/MS) analysis was performed to identify the possible radioprotectors. In vivo and in vitro studies, namely Real-Time-PCR (RT-PCR) analysis, Reactive Oxygen Species (ROS) scavenging activity, lipid peroxidation and GSH levels, DNA damage and cell death studies, anti-inflammatory (Sandwich ELISA), immunomodulatory studies (antibody staining), and model free radical scavenging assays, were performed. Vanillic acid, protocatechuic acid, para-hydroxybenzoic acid, chlorogenic acid, TNF-α inhibitor (Eudesmin), isoflavone (Daidzein 7-o-glucoside), astragalin (Kaempferol 3-o-glycoside), and other polyphenols were identified in PSHE using UHPLC-HRMS/MS analysis. Prophylactic administration of PSHE (−1 h) rendered more than 33% survival in mice exposed to 8 Gy whole-body-irradiation with increased mice survival and recovery of bone marrow and spleen cellularity. Real-time RT-PCR analysis showed that PSHE treatment (50 µg/mL) upregulated Nrf2, HO-1, and GPX-1 in mice splenocytes. At 50 µg/mL, PSHE reduced ROSscavenging activity, mitochondrial and spleen membrane lipid peroxidation levels, DNA damage, and cell death, and increased GSH levels. At 10 µg/mL, PSHE treatment diminished the content of IL-6 and TNF-α. At 50 µg/mL, PSHE suppressed lymphocyte proliferation. These findings indicate that polyphenols of PSHE possess marked antioxidant, anti-inflammatory, and immunomodulatory capacities, which play important roles in the prevention of radiation damage.

A data-independent acquisition approach based on HRMS to explore the biodegradation process of organic micropollutants involved in a biological ion-exchange drinking water filter

Drinking water producers continuously develop innovative treatment processes to effectively remove organic micropollutants from raw water. Biological ion-exchange (BIEX) water treatment is one of these new techniques under development and showing great potential. In order to investigate if biodegradation is highly involved in such a removal technique, cultures were prepared with microorganisms sampled on the resins of a BIEX filter. Then, organic micropollutants were spiked into these cultures and their (bio)degradation was followed over 30 days by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The purpose of this study was firstly to develop an analytical method using UHPLC-HRMS able to monitor the degradation of three spiked organic micropollutants in culture. Beyond quantification, this method allowed the simultaneous recording of fragmentation information via the use of a data-independent acquisition approach to perform a non-exhaustive search of transformation products related to the spiked micropollutants in culture aliquots. Secondly, a data treatment approach was developed to process raw spectral data generated by aliquots analysis by optimizing the precursor isolation mass windows, the accurate mass tolerance, peak intensity thresholds and choice of database. The use of this new method with a post-data acquisition treatment approach completed by the exhaustive study of fragmentation spectra allowed the tentative identification of 11 transformation products related to the spiked compounds. Finally, 16S rRNA gene amplicon sequencing revealed that bacterial genera known for their ability to degrade the spiked micropollutants were present in the microbial community of the BIEX drinking water filter.

Blood plasma metabolomics of children and adolescents with sickle cell anaemia treated with hydroxycarbamide: a new tool for uncovering biochemical alterations.

Sickle cell anaemia (SCA) is a debilitating genetic haemoglobinopathy predominantly affecting the disenfranchised strata of society in Africa and the Americas. The most common pharmacological treatment for this disease is the administration of hydroxycarbamide (HC) for which questions remain regarding its mechanism of action, efficacy and long-term toxicity specifically in paediatric individuals. A multiplatform metabolomics approach was used to assess the metabolome of plasma samples from a population of children and adolescents with SCA with and without HC treatment along with non-SCA individuals. Fifty-three metabolites were identified by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) and 1 H nuclear magnetic resonance (NMR) with a predominance of membrane lipids, amino acids and organic acids. The partial least-squares discriminant analysis (PLS-DA) analysis allowed a clear discrimination between the different studied groups, revealing clear effects of the HC treatment in the patients' metabolome including rescue of specific metabolites to control levels. Increased creatine/creatinine levels under HC treatment suggests a possible increase in the arginine pool and increased NO synthesis, supporting existing models for HC action in SCA. The metabolomics results extend the current knowledge on the models for SCA pathophysiology including impairment of Lands' cycle and increased synthesis of sphingosine 1-phosphate. Putative novel biomarkers are suggested.

The glycosyltransferase UGT76B1 modulates N-hydroxy-pipecolic acid homeostasis and plant immunity.

The tradeoff between growth and defense is a critical aspect of plant immunity. Therefore, the plant immune response needs to be tightly regulated. Salicylic acid (SA) is an important plant hormone regulating defense against biotrophic pathogens. Recently, N-hydroxy-pipecolic acid (NHP) was identified as another regulator for plant innate immunity and systemic acquired resistance (SAR). Although the biosynthetic pathway leading to NHP formation is already been identified, how NHP is further metabolized is unclear. Here, we present UGT76B1 as a uridine diphosphate-dependent glycosyltransferase (UGT) that modifies NHP by catalyzing the formation of 1-O-glucosyl-pipecolic acid in Arabidopsis thaliana. Analysis of T-DNA and clustered regularly interspaced short palindromic repeats (CRISPR) knock-out mutant lines of UGT76B1 by targeted and nontargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) underlined NHP and SA as endogenous substrates of this enzyme in response to Pseudomonas infection and UV treatment. ugt76b1 mutant plants have a dwarf phenotype and constitutive defense response which can be suppressed by loss of function of the NHP biosynthetic enzyme FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). This suggests that elevated accumulation of NHP contributes to the enhanced disease resistance in ugt76b1. Externally applied NHP can move to distal tissue in ugt76b1 mutant plants. Although glycosylation is not required for the long-distance movement of NHP during SAR, it is crucial to balance growth and defense.