Longitudinal Plant Health Monitoring via High-Resolution Mass Spectrometry Screening Workflows: Application to a Fertilizer Mediated Tomato Growth Experiment.

Significant efforts have been spent in the modern era towards implementing environmentally friendly procedures like composting to mitigate the negative effects of intensive agricultural practices. In this context, a novel fertilizer was produced via the hydrolysis of an onion-derived compost, and has been previously comprehensively chemically characterized. In order to characterize its efficacy, the product was applied to tomato plants at five time points to monitor plant health and growth. Control samples were also used at each time point to eliminate confounding parameters due to the plant's normal growth process. After harvesting, the plant leaves were extracted using aq. MeOH (70:30, v/v) and analyzed via UPLC-QToF-MS, using a C18 column in both ionization modes (±ESI). The data-independent (DIA/bbCID) acquisition mode was employed, and the data were analyzed by MS-DIAL. Statistical analysis, including multivariate and trend analysis for longitudinal monitoring, were employed to highlight the differentiated features among the controls and treated plants as well as the time-point sequence. Metabolites related to plant growth belonging to several chemical classes were identified, proving the efficacy of the fertilizer product. Furthermore, the efficiency of the analytical and statistical workflows utilized was demonstrated.

Comprehensive HRMS Chemical Characterization of Pomegranate-Based Antioxidant Drinks via a Newly Developed Suspect and Target Screening Workflow.

Antioxidants play a significant role in human health, protecting against a variety of diseases. Therefore, the development of products with antioxidant activity is becoming increasingly prominent in the human lifestyle. New antioxidant drinks containing different percentages of pomegranate, blackberries, red grapes, and aronia have been designed, developed, and manufactured by a local industry. The comprehensive characterization of the drinks' constituents has been deemed necessary to evaluate their bioactivity. Thus, LC-qTOFMS has been selected, due to its sensitivity and structure identification capability. Both data-dependent and -independent acquisition modes have been utilized. The data have been treated according to a novel, newly designed workflow based on MS-DIAL and MZmine for suspect, as well as target screening. The classical MS-DIAL workflow has been modified to perform suspect and target screening in an automatic way. Furthermore, a novel methodology based on a compiled bioactivity-driven suspect list was developed and expanded with combinatorial enumeration to include metabolism products of the highlighted metabolites. Compounds belonging to ontologies with possible antioxidant capacity have been identified, such as flavonoids, amino acids, and fatty acids, which could be beneficial to human health, revealing the importance of the produced drinks as well as the efficacy of the new in-house developed workflow.

Complete chemical characterization of Crocus sativus via LC-HRMS: Does trimming affect the chemical content of saffron?

Saffron, a spice derived from Crocus sativus, which in Iran is subjected to different trimming, is known for its beneficial health effects and high market value. Authentication studies related to geographical origin and adulterants presence mainly exist in literature, however fraud due to trimming has not been reported. In the current research, chemical characterization of six saffron trims, namely Sargol, Negin, Pushal, Bunch, Style, and Powder, was accomplished through suspect and non-target screening employing LC-QToF-MS in both electrospray ionization modes. The samples were extracted using methanol:water (50:50,v:v) and 62 compounds were identified, including amino acids, vitamins, flavonoids, phenolics, carotenoids, cyclohexenones. A clear discrimination among the red trims (Pushal, Sargol and Negin), as well as between Style and Bunch using Multivariate Chemometrics techniques was achieved. Proline and isophorone were highlighted as authenticity markers. Finally, the effect of three harvesting year on the most contributing compounds for trimming discrimination has been evaluated.

Two Fast GC-MS Methods for the Measurement of Nicotine, Propylene Glycol, Vegetable Glycol, Ethylmaltol, Diacetyl, and Acetylpropionyl in Refill Liquids for E-Cigarettes.

The use of e-cigarettes (ECs) has become increasingly popular worldwide, even though scientific results have not established their safety. Diacetyl (DA) and acetylpropionyl (AP), which can be present in ECs, are linked with lung diseases. Ethyl maltol (EM)-the most commonly used flavoring agent-can be present in toxic concentrations. Until now, there is no methodology for the determination of nicotine, propylene glycol (PG), vegetable glycerin (VG), EM, DA, and acetylpropionyl in e-liquids that can be used as a quality control procedure. Herein, gas chromatography coupled with mass spectrometry (GC-MS) was applied for the development of analytical methodologies for these substances. Two GC-MS methodologies were developed and fully validated, fulfilling the standards for the integration in a routine quality control procedure by manufacturers. As proof of applicability, the methodology was applied for the analysis of several e-liquids. Differences were observed between the labeled and the experimental levels of PG, VG, and nicotine. Three samples contained EM at higher concentrations compared to the other samples, while only one contained DA. These validated methodologies can be used for the quality control analysis of EC liquid samples regarding nicotine, PG, and VG amounts, as well as for the measurement of the EM.

From By-Products to Fertilizer: Chemical Characterization Using UPLC-QToF-MS via Suspect and Non-Target Screening Strategies.

The increasing demands of agriculture and the food market have resulted in intensive agricultural practices using synthetic fertilizers to maximize production. However, significant efforts have been made to implement more environmentally friendly procedures, such as composting, to overcome the adverse impact of these invasive practices. In the terms of this research, composting was applied to the production of two biofertilizers, using onion and mushroom by-products as raw materials respectively. The main purposes of this work were to identify the compounds that pass from the raw materials to the final products (onion-based and mushroom-based), as well as the characterization of the chemical profile of these final products following suspect and non-target screening workflows via UPLC-qToF-MS. Overall, 14 common compounds were identified in the onion and its final product, while 12 compounds were found in the mushroom and its corresponding product. These compounds belong to fatty acids, organic acids, and flavonoids, which could be beneficial to plant health. The determination of parameters, such as the pH, conductivity, organic matter, nitrogen content, and elemental analysis, were conducted for the overall characterization of the aforementioned products.

Plasma Metabolomic Alterations Induced by COVID-19 Vaccination Reveal Putative Biomarkers Reflecting the Immune Response.

Vaccination is currently the most effective strategy for the mitigation of the COVID-19 pandemic. mRNA vaccines trigger the immune system to produce neutralizing antibodies (NAbs) against SARS-CoV-2 spike proteins. However, the underlying molecular processes affecting immune response after vaccination remain poorly understood, while there is significant heterogeneity in the immune response among individuals. Metabolomics have often been used to provide a deeper understanding of immune cell responses, but in the context of COVID-19 vaccination such data are scarce. Mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR)-based metabolomics were used to provide insights based on the baseline metabolic profile and metabolic alterations induced after mRNA vaccination in paired blood plasma samples collected and analysed before the first and second vaccination and at 3 months post first dose. Based on the level of NAbs just before the second dose, two groups, "low" and "high" responders, were defined. Distinct plasma metabolic profiles were observed in relation to the level of immune response, highlighting the role of amino acid metabolism and the lipid profile as predictive markers of response to vaccination. Furthermore, levels of plasma ceramides along with certain amino acids could emerge as predictive biomarkers of response and severity of inflammation.

Effect of Supplementation with Olive Leaf Extract Enriched with Oleuropein on the Metabolome and Redox Status of Athletes' Blood and Urine-A Metabolomic Approach.

Oleuropein (OE) is a secoiridoid glycoside occurring mostly in the Oleaceae family and presenting several pharmacological properties, including hypolipidemic and antioxidant properties. Based on these, several dietary supplements containing olive leaf extracts enriched with OE are commercially available in many countries. The current study aimed to examine the effect of supplementation with such an extract on the serum and urine metabolome of young healthy male athletes. For this purpose, applying a randomized, balanced, double-blind study, nine young, healthy males (physical education students) received either a commercially prepared extract or placebo for one week, followed by a two-week washout period; then, they were subsequently dosed with the alternate scheme (crossover design). Urine and serum samples were analyzed using UHPLC-HRMS, followed by evaluation with several multivariate methods of data analysis. The data were interpreted using a multilevel metabolomic approach (multilevel-sPLSDA) as it was found to be the most efficient approach for the study design. Metabolic pathway analysis of the most affected metabolites revealed that tryptophan and acylcarnitine's biochemistries were most influenced. Furthermore, several metabolites connected to indole metabolism were detected, which may indicate enhanced serotonin turnover. Phenylethylamine and related metabolites, as well as estrone, were connected to enhanced performance. In addition, possible changes to the lipidemic profile and the blood and urine redox statuses were investigated.

Targeted Metabolomics: The LC-MS/MS Based Quantification of the Metabolites Involved in the Methylation Biochemical Pathways.

Biochemical methylation reactions mediate the transfer of the methyl group regulating vital biochemical reactions implicated in various diseases as well as the methylation of DNA regulating the replication processes occurring in living organisms. As a finite number of methyl carriers are involved in the methyl transfer, their quantification could aid towards the assessment of an organism's methylation potential. An Hydrophilic Interaction Chromatography-Liquid Chromatography Multiple Reaction Monitoring (HILIC-LC-MRM) mass spectrometry (MS) methodology was developed and validated according to Food & Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) for the simultaneous determination of nine metabolites i.e., B12, folic acid, 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, betaine, phosphocholine, N,N-dimethylglycine, and deoxythymidine monophosphate in human blood plasma. The sample pretreatment was based on a single step Solid-phase extraction (SPE) methodology using C18 cartridges. The methodology was found to accurately quantitate the analytes under investigation according to the corresponding dynamic range proposed in the literature for each analyte. The applicability of the method was assessed using blood donor samples and its applicability demonstrated by the assessment of their basal levels, which were shown to agree with the established basal levels. The methodology can be used for diagnostic purposes as well as for epigenetic screening.

A Novel Validated Injectable Colistimethate Sodium Analysis Combining Advanced Chemometrics and Design of Experiments.

Colistimethate sodium (CMS) is widely administrated for the treatment of life-threatening infections caused by multidrug-resistant Gram-negative bacteria. Until now, the quality control of CMS formulations has been based on microbiological assays. Herein, an ultra-high-performance liquid chromatography coupled to ultraviolet detector methodology was developed for the quantitation of CMS in injectable formulations. The design of experiments was performed for the optimization of the chromatographic parameters. The chromatographic separation was achieved using a Waters Acquity BEH C8 column employing gradient elution with a mobile phase consisting of (A) 0.001 M aq. ammonium formate and (B) methanol/acetonitrile 79/21 (v/v). CMS compounds were detected at 214 nm. In all, 23 univariate linear-regression models were constructed to measure CMS compounds separately, and one partial least-square regression (PLSr) model constructed to assess the total CMS amount in formulations. The method was validated over the range 100-220 µg mL-1. The developed methodology was employed to analyze several batches of CMS injectable formulations that were also compared against a reference batch employing a Principal Component Analysis, similarity and distance measures, heatmaps and the structural similarity index. The methodology was based on freely available software in order to be readily available for the pharmaceutical industry.

Colistimethate Acidic Hydrolysis Revisited: Arrhenius Equation Modeling Using UPLC-QToF MS.

Colistimethate (CMS), the prodrug of polymyxin E (colistin), is an antibiotic widely used as a last-line therapy against multidrug resistant Gram-negative bacteria, but little is known about its pharmacokinetics as its administration has stopped as a result of high neuro- and nephro-toxicity. The measurement of CMS levels in patients' biological fluids is of great importance in order to find the optimal dose regimen reducing the drug toxicity. Until now, CMS assay methods are based on the indirect determination after its hydrolysis to colistin (CS). Herein, the aim is to find the optimal conditions for the complete hydrolysis of CMS to CS. The reaction was studied at accelerated conditions: 40 °C, 50 °C, and 60 °C, and the results were evaluated by assessing the Arrhenius equation and computation employing the Tenua software. A validated analytical methodology based on ultra-performance liquid chromatography (UPLC) coupled to a hybrid quadrupole time of flight (QToF) instrument is developed for the simultaneous measurement of CMS and CS. The current methodology resulted in complete hydrolysis, in contrast with the previously reported one.

Design of experiments guided multivariate calibration for the quantitation of injectable colistimethate sodium by ultra performance liquid chromatography - High resolution mass spectrometry.

Colistimethate sodium (CMS) is a widely administrated old-generation prodrug for the treatment of the life-threatening infections caused by multi-resistant Gram-negative bacteria. Until now, the quality control procedure of the CMS commercial products is based on microbiological assays. The aim of the study is the development of a chemical analysis methodology based on liquid chromatography - mass spectrometry (LC-MS) that could be used for the quality control of CMS products. The careful optimization of the LC and QToF-MS parameters was deemed crucial, as CMS is known to be a very complex mixture. Thus, a two stage Design of Experiments (DoE) pipeline has been followed, aiming towards the separation of the mixture components. According to the DoE results, a baseline-resolved chromatogram revealing more than 20 compounds was achieved. The separation was performed using a Waters Acquity BEH C8 column employing gradient elution. The mobile phase consisted of aq. ammonium formate 0.005 M (pH 6) (solvent A) and methanol/acetonitrile 79/21 (v/v) (solvent B). A second optimization experiment for the MS signal was employed in order to achieve maximum sensitivity. The singly charged signals were monitored for the validation in the positive ion mode. The calibration curve range was 50-110 µg mL-1, corresponding to the 80-120% of the nominal CMS amount in the commercial products. Due to the complexity of the CMS chromatograms and the corresponding spectrum of each chromatographic peak, untargeted and targeted approaches were performed employing the MZmine software. Furthermore, apart from the classical univariate statistical analysis, partial least squares regression (PLS-R) model was also employed, as the variables were more than the observations. The developed methodology has been employed to analyze several batches and inconsistences have been discovered.

A multi-technique analytical approach for impurity profiling during synthesis: The case of difluprednate.

A methodology for the qualitative analysis of a mixture of compounds obtained during the synthesis of difluprednate is described herein for the first time. For this scope a multi-technique analytical approach was developed, combining Liquid Chromatography/Mass Spectrometry (LC/MS), Nuclear Magnetic Resonance (NMR) and computational chemistry. Separation of isomers is frequently required for the identification of impurities in active pharmaceutical ingredients (APIs) to assess the impact they may exhibit on public health. During the final step of the difluprednate synthesis apart from the desired product, various by-products may be obtained. Structural analysis of the products using LC/MS and NMR indicated that the steroid difluprednate was obtained along with its acetyl/butyryl regional isomers, whereas the results were further supported by semi-empirical calculations of the MS-derived data. Following the proposed approach, we managed to elucidate the structures of the challenging 11-acetate, 17-butyrate from the 17-acetate, 21-butyrate, 6α,9α-difluoro prednisolone isomers. The approach utilized may be of general applicability for the analysis of impurities in active pharmaceutical ingredients obtained during chemical synthesis.

Human Melanoma-Cell Metabolic Profiling: Identification of Novel Biomarkers Indicating Metastasis.

Melanoma is the most aggressive type of skin cancer, leading to metabolic rewiring and enhancement of metastatic transformation. Efforts to improve its early and accurate diagnosis are largely based on preclinical models and especially cell lines. Hence, we herein present a combinational Nuclear Magnetic Resonance (NMR)- and Ultra High Performance Liquid Chromatography-High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS)-mediated untargeted metabolomic profiling of melanoma cells, to landscape metabolic alterations likely controlling metastasis. The cell lines WM115 and WM2664, which belong to the same patient, were examined, with WM115 being derived from a primary, pre-metastatic, tumor and WM2664 clonally expanded from lymph-node metastases. Metabolite samples were analyzed using NMR and UHPLC-HRMS. Multivariate statistical analysis of high resolution NMR and MS (positive and negative ionization) results was performed by Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), while metastasis-related biomarkers were determined on the basis of VIP lists, S-plots and Student's t-tests. Receiver Operating Characteristic (ROC) curves of NMR and MS data revealed significantly differentiated metabolite profiles for each cell line, with WM115 being mainly characterized by upregulated levels of phosphocholine, choline, guanosine and inosine. Interestingly, WM2664 showed notably increased contents of hypoxanthine, myo-inositol, glutamic acid, organic acids, purines, pyrimidines, AMP, ADP, ATP and UDP(s), thus indicating the critical roles of purine, pyrimidine and amino acid metabolism during human melanoma metastasis.

Malignancy Grade-Dependent Mapping of Metabolic Landscapes in Human Urothelial Bladder Cancer: Identification of Novel, Diagnostic, and Druggable Biomarkers.

BACKGROUND: Urothelial bladder cancer (UBC) is one of the cancers with the highest mortality rate and prevalence worldwide; however, the clinical management of the disease remains challenging. Metabolomics has emerged as a powerful tool with beneficial applications in cancer biology and thus can provide new insights on the underlying mechanisms of UBC progression and/or reveal novel diagnostic and therapeutic schemes. METHODS: A collection of four human UBC cell lines that critically reflect the different malignancy grades of UBC was employed; RT4 (grade I), RT112 (grade II), T24 (grade III), and TCCSUP (grade IV). They were examined using Nuclear Magnetic Resonance, Mass Spectrometry, and advanced statistical approaches, with the goal of creating new metabolic profiles that are mechanistically associated with UBC progression toward metastasis. RESULTS: Distinct metabolic profiles were observed for each cell line group, with T24 (grade III) cells exhibiting the most abundant metabolite contents. AMP and creatine phosphate were highly increased in the T24 cell line compared to the RT4 (grade I) cell line, indicating the major energetic transformation to which UBC cells are being subjected during metastasis. Thymosin ß4 and ß10 were also profiled with grade-specific patterns of expression, strongly suggesting the importance of actin-cytoskeleton dynamics for UBC advancement to metastatic and drug-tolerant forms. CONCLUSIONS: The present study unveils a novel and putatively druggable metabolic signature that holds strong promise for early diagnosis and the successful chemotherapy of UBC disease.

A novel UHPLC-HRMS-based metabolomics strategy enables the discovery of potential neuroactive metabolites in mice plasma, following i.p. administration of the main Crocus sativus L. bioactive component.

Trans-crocin 4 (TC4) is an important carotenoid constituent of saffron showing potential activity against Alzheimer's Disease (AD) due to its antioxidant and antiamyloidogenic properties. Metabolomics is an emerging scientific field that enhances biomarker discovery and reveals underlying biochemical mechanisms aiming towards the early subclinical diagnosis of diseases. To date, there are no reports on the changes induced to mice plasma metabolome after TC4 administration. We report a novel untargeted UHPLC-ESI HRMS metabolomics strategy to determine the alteration of the metabolic fingerprint following i.p. administration of TC4 in male and female mice. Blood samples from fiftysix mice treated with TC4 as well as from control animals were analyzed with UHPLC-ESI HRMS. Statistical evaluation of the results was achieved by multivariate analysis (MVA), i.e., principal component analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) in order to discover the variables that contributed to the discrimination between treated and untreated groups which were identified by online database searching (e.g., Metlin, HMDB, KEGG) aided by chemometric processing, e.g., covariance searching etc. Due to the high variability imposed by various factors, e.g., sex of the animals participating in the study, administration dose and time-points of sacrifice, multilevel sparse PLS-DA analysis, e.g., splitting variation to each individual component, has been employed as a more efficient approach for such designs. This methodology allowed the identification of the time sequence of metabolome changes due to the administration of TC4, whereas a sex-related effect on the metabolome is indicated, denoting that the administration in both sexes is indispensable in order to acquire safe conclusions as reliable metabolome pictures. The results demonstrated a number of annotated metabolites playing a potential role in neuroprotection while they are closely related to AD. Moreover, five additional annotated metabolites were involved in the steroid biosynthesis pathway while two of them may be considered as putative neuroprotective agents.

Development and Validation of a UPLC-ESI(-)-MS/MS Methodology for the Simultaneous Quantification of Hesperidin, Naringin, and their Aglycones in Chicken Tissue Samples.

BACKGROUND: The dietary supplementation of livestock with antioxidants to improve the meat quality represents an active research area of high commercial impact. In order to investigate the optimal dosing, analytical methodologies need to be developed in various tissues to evaluate which concentration does remain in the tissue. OBJECTIVE: We aimed to develop and validate a sensitive and specific methodology for the simultaneous quantitative determination of hesperidin, naringin, hesperetin, and naringenin in chicken tissue samples employing ultra-performance LC-tandem MS. METHODS: Lipid extraction using cold chloroform was performed followed by protein precipitation by cold acetone. Chromatography was performed on a C18 column using a ternary gradient of water, acetonitrile, and isopropanol-acetonitrile-acetone (58+40+2, v/v) as the mobile phase. Detection was performed by electrospray ionization in negative ion mode with the selected reaction monitoring technique. RESULTS: Calibration plots exhibited good linearity (r2 > 0.99) over the concentration range from 0.125 to 25 µg/g tissue for the four analytes, and the lower LOQ for the four analytes was 0.125 µg/g tissue. The repeatability as percent relative SD and precision as percent accuracy were <20 and >80%, respectively. CONCLUSIONS: The developed methodology was applied for the quantitative determination of hesperidin, naringin, hesperetin, and naringenin in tissue samples after dietary supplementation with 1.5 g/kg hesperidin and 1.5 g/kg naringin in Ross 308 broiler chickens. HIGHLIGHTS: This is the first methodology to access naringin, naringenin, hesperidin, and hesperetin in chicken tissue. It involved simple sample preparation, and the mass spectrometry based detection ensures high specificity and sensitivity.

Analytical methodologies used for the determination of colistin in biological fluids. Is it still a challenge?

Colistin is a multicomponent polypeptide antibiotic consisting mainly of colistin A and colistin B, produced by selected strains of Bacillus polymyxa var. Colistinus. Only recently, the prodrug of colistin, colistimethate sodium, is widely used as last resort antibiotic for infections caused by resistant gram-negative bacteria. Colistin having been discovered several years ago, has not subjected to the drug development and regulatory approval processes that are applied today. However, pharmacological and pharmacokinetic information are necessary for its optimal use thus, during the last decades several studies are carried out in order to shed light on this issue. In the current review, the analytical methodologies of colistin assessment in biological material are summarized and the analytical challenges are critically discussed and critical aspects of the determinations such as the method of detection, the sample pretreatment methodology etc. are compared. Furthermore, critical quality aspects of the assessment methodologies such as the sensitivity of the currently developed methodologies are presented. Lastly, some future trends that should be incorporated in the determination pipeline of modern drugs are suggested.

UHPLC-HRMS-based tissue untargeted metabolomics study of naringin and hesperidin after dietary supplementation in chickens.

To date numerous metabolomic studies have been performed in order to characterize nutritional intervention studies. The aim of the current study was to present a comprehensive pipeline for characterizing the metabolic changes that occur in chickens tissues in response to naringin and hesperidin dietary supplementation. Forty-nine chickens were randomly divided into 3 groups: the first one fed with diet supplemented with naringin, the second with hesperidin whereas the control group was fed by commercial basal diet. After 30 days of administration chicken muscle samples were analyzed by UHPLC-HRMS whereas data were analyzed by the proposed pipeline. Three significant variables were detected to discriminate the control from the group after naringin administration and thirteen variables after hesperidin supplementation. Furthermore, a more detailed pipeline (encompassing multiple internal standards, internal validation of the clustering, extended statistical significance scores and multiple identification procedures) has been proposed aiming towards a more accurate untargeted analysis.

Trans-crocin 4 is not hydrolyzed to crocetin following i.p. administration in mice, while it shows penetration through the blood brain barrier.

A novel, fit-for-purpose, highly sensitive, analytical UPLC-PDA methodology was developed and fully validated, according to ICH, FDA and EMA guidelines, for the rapid and accurate quantification of trans-crocin 4 (TC4) and crocetin (CRC) in mice plasma and brain after i.p. administration. A PDA based methodology shows a wider applicability as it is cost effective and can be easily and seamlessly adopted by the pharma industry. The separation of the analytes was performed on a C18 Hypersil Gold column with 2.5 min run time, employing the internal standard (ISTD) methodology. The two methods were successfully applied for the determination of CRC and TC4 in mouse plasma and brain after i.p. administration of TC4 (50 mg/kg) in a time range of 0-240 min. Due to the selection of i.p. administration route, the first-pass metabolism and/or gastric hydrolysis were bypassed, a fact that enhanced the bioavailability of TC4. Furthermore, TC4 was found to be capable of crossing the Blood Brain Barrier (BBB) and build up levels in the mouse brain, regardless of its highly hydrophilic character. CRC was not detected in any plasma or brain sample, although it has been reported that TC4 quickly hydrolyzes to CRC after p.o. administration. Therefore i.p. administration could be used in the case of TC4 for the accurate determination of its biological role. Overall, the developed methodology offers important information about the bioavailability of TC4 in mouse plasma and for the first time, demonstrates the ability of TC4 to penetrate the BBB and localize inside the brain.

Alteration in the liver metabolome of rats with metabolic syndrome after treatment with Hydroxytyrosol. A Mass Spectrometry And Nuclear Magnetic Resonance - based metabolomics study.

Metabolic syndrome (MetS) represents a group of abnormalities that enhances the risk for cardiovascular disease, diabetes and stroke. The Mediterranean diet seems to be an important dietary pattern, which reduces the incidence of MetS. Hydroxytyrosol (HT) - a simple phenol found in olive oil - has received increased attention for its antioxidant activity. Recently, the European Foods Safety Authority (EFSA) claimed that dietary consumption of HT exhibits a protective role against cardiovascular disease. In this study, an experimental protocol has been setup, including isolated HT administration in a diet induced model of MetS in young Wistar rats, in order to find out whether HT has a protective effect against MetS. Rats were randomly divided into two groups nurtured by high-carbohydrate high-fat (H) (MetS inducing diet) and high-carbohydrate high-fat + HT (HHT). HT (20mg/kg/d oral gavage, water vehicle) was administered for 8 weeks on the basal diet. Previous pharmacological evaluation of HT showed that hepatic steatosis was reduced and the inflammatory cells into the liver were infiltrated. These indicate that HT shows bioactivity against metabolic syndrome. Therefore, the metabolomics evaluation of liver extracts would indicate the putative biochemical mechanisms of HT activity. Thus, the extracts of liver tissues were analyzed using Ultra Performance Liquid Chromatography - High Resolution Mass Spectrometry (UPLC-HRMS, Orbitrap Discovery) and Nuclear Magnetic Resonance (NMR) spectroscopy (Bruker Avance III 600MHz). Multivariate analysis was performed in order to gain insight on the metabolic effects of HT administration on the liver metabolome. Normalization employing multiple internal standards and Quality Control-based Robust LOESS (LOcally Estimated Scatterplot Smoothing) Signal Correction algorithm (QC-RLSC) was added in the processing pipeline to enhance the reliability of metabolomic analysis by reducing unwanted information. Experimentally, HHT rats were clearly distinguished from H in PLS-DA, showing differences in the liver metabolome between the groups and specific biomarkers were determined supporting the pharmacological findings. More specifically, HT has shown to be effective towards the mobilization of lipids as various lipid classes being differentially regulated between the H and HHT groups. Interestingly branched fatty acid esters of hydroxy oleic acids (OAHSA) lipids have been shown to be up regulated to the HHT group, denoting the alleviation of the MetS to the animals administered with HT.