Recently the number of studies investigating triterpenoid saponins has drastically increased due to their diverse and potentially attractive biological activities. Currently the literature contains chemical structures of few hundreds of triterpenoid saponins of plant and animal origin. Triterpenoid saponins consist of a triterpene aglycone with one or more sugar moieties attached to it. However, due to similar physico-chemical properties, isolation and identification of a large diversity of triterpenoid saponins remain challenging. This study demonstrates a methodology to screen saponins using hyphenated analytical platforms, GC-MS, LC-MS/MS, and LC-SPE-NMR/MS, in the example of two different phenotypes of the model plant Barbarea vulgaris (winter cress), glabrous (G) and pubescent (P) type that are known to differ by their insect resistance. The proposed methodology allows for detailed comparison of saponin profiles from intact plant extracts as well as saponin aglycone profiles from hydrolysed samples. Continuously measured 1D proton NMR data during LC separation along with mass spectrometry data revealed significant differences, including contents of saponins, types of aglycones and numbers of sugar moieties attached to the aglycone. A total of 49 peaks were tentatively identified as saponins from both plants; they are derived from eight types of aglycones and with 2–5 sugar moieties. Identification of two previously known insect-deterrent saponins, hederagenin cellobioside and oleanolic acid cellobioside, demonstrated the applicability of the methodology for relatively rapid screening of bioactive compounds.

Background: We prepared reference materials of fentanyl and amphetamines in hair, and established methods for the certification of reference materials of fentanyl and amphetamines in hair samples. Methods: Hair samples containing 15 drugs were prepared by immersion method (water-dimethylsulfoxide). On the basis of single-factor experiment, Box-Behnken design-response surface was used to optimize the pre-treatment conditions of samples. In MRM/SIM mode, 15 drugs were quantitatively analyzed by LC-MS/MS and GC-MS/MS methods. Results: The contents of 15 drugs in the mixed hair samples were: 1.050 ng mg-1~7.771 ng mg-1, RSDs<7%(n=11). The results showed that the two analytical methods developed were highly sensitive (LODs as low as 0.05 pg mg-1), reproducible (intra-day and inter-day precision RSDs were<10%) and accurate. F-test was performed on the prepared hair samples and the results showed that the homogeneity of the prepared hair samples was good. Conclusions: The established methods were used for the detection of fentanyl and amphetamine drugs in human hair, with high sensitivity, accuracy and specificity. It can be used as a certified method for the development of fentanyl and amphetamine matrix certified reference materials. The developed hair reference materials are expected to be applied in forensic drug abuse detection and laboratory quality control.

NH4Cl is one of the nitrogen sources for microalgal cultivation. However, excessive amounts of NH4Cl affects microalgal physiology and biomass contents. In this study, the effects of ammonium on microalgal growth and TAG content in the green microalga (Chlamydomonas reinhardtii) was investigated. Microalgal growth and TAG content under photoautotrophic conditions were found to be unchanged with 17 mM of ammonium, while this compound interfered with microalgal growth and induced TAG content under mixotrophic conditions with acetate supplementation. This suggested that ammonium could induce TAG production when acetate occurred in microalgal cultivation. Further, the effects of two different concentrations of NH4Cl (17 mM and 60 mM) on the cells under mixotrophic conditions were investigated. The results showed that both concentrations reduced microalgal growth, but induced total lipid and TAG content, especially after a 4-day cultivation. The oxygen evolution and Fv/Fm ratio showed that both concentrations completely inhibited the oxygen evolution on Day 4. The 60 mM NH4Cl reduced the Fv/Fm ratio from 0.7 to 0.48 indicating that ammonium supplementation directly affects the microalgae photosynthesis performance. A total of 1782 proteins were successfully identified using proteomics analysis. Among them, there were nine overexpressed proteins and four proteins were underexpressed. Using the protein–ligand interaction analysis, nitrogen metabolism is involved under NH4Cl conditions. This information can provide biochemical knowledge for microalgae development for sustainable energy usage.

Background and objectives: Lipoid proteinosis (LP) is an autosomal recessive transfer lysosomal storage disease, characterised by the accumulation of hyalin substance in the mucous membranes, skin, internal organs and brain, for which there is no biochemical diagnostic method. The aim of this study was to determine the carnitine and acylcarnitine metabolic profile with LC-MS/MS in LP patients and thereby examine the potential of this as a new biochemical method in the determination of biochemical markers in LP patients. Materials and Methods: In this study, 27 carnitine and acylcarnitine esters were measured with LC-MS/MS in serum samples taken from 14 healthy control subjects and 14 patients who presented at the Skin and Venereal Diseases Polyclinic and were diagnosed with LP as a result of clinical, radiological and histopathological examinations. Results: The results of the study showed that C0 (free carnitine) C3, C4, C4:DC, C5DC, C6, C8, C14:1, C14:2, C16 and C18 acylcarnitines were statistically significantly reduced in the LP patients (p<0.05, p<0.01). Conclusions: It was concluded that the application of carnitine profile screening, which is an inexpensive, rapid and reliable method, could make a contribution to the differential diagnosis as aa supporting laboratory test in individuals with suspected LP.

In the last decade, the tremendous improvement in the sensitivity and also affordability of Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) have revolutionized its application in pharmaceutical analysis, resulting in wide-spread of employing LC-MS/MS for determining pharmaceutical compounds including anticancer drugs in pharmaceutical research and also industries. Currently, LC-MS/MS has been widely used to quantify small molecule oncology drugs in various biological matrices to support preclinical and clinical Pharmacokinetic studies in R & D of oncology drugs. This mini-review article will describe the state-of-the art LC-MS/MS and its application in rapid quantification of small molecule anticancer drugs. In addition, efforts have also been made in this review to address several key aspects in the development of rapid LC-MS/MS methods, including sample preparation, chromatographic separation and matrix effect evaluation.

A sensitive and robust liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of nimbolide in mouse serum. Exemestane was used as the internal standard (IS). Here, we employed acetonitrile-based protein precipitation (PPT) for serum sample preparation, and performed chromatographic separation using an ODS Hypersil C18 column (100×2.1 mm, 5µm) with gradient elution (0.1% formic acid in water vs 100% acetonitrile). The run time was 6 min. Instrumental analysis was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the multiple-reaction monitoring (MRM) under positive mode. A good linear calibration was achieved in the 5–1000 ng/ml range. The intra- and inter-day precisions for nimbolide were ≤ 12.6% and ≤ 13.9 % respectively. Intra-day accuracy ranged from 96.9% – 109.3% while inter-day accuracy ranged from 94.3% – 110.2%. The matrix effect of nimbolide, detected but consistent at low and high concentrations, do not affect linearity of standard curve. In conclusion, we have developed and validated a sensitive analytical method for determination of a novel natural compound nimbolide in mouse serum and it has been successfully applied to our preclinical study in investigating the pharmacokinetic properties of nimbolide, which could greatly facilitate the preclinical development of the promising lead compound for anticancer therapy.

BPA is a commonly used compound in many industries and has versatile applications in polycarbonate plastics and epoxy resins production. BPA is classified as endocrine-disrupting chemical which can hamper fetal development during pregnancy and may have long term negative health outcomes in humans. Dietary sources, main route of BPA exposure, can be contaminated by the migration of BPA into food during processing. The global regulatory framework for using this compound in food contact materials is currently not harmonized. This review aims to outline, survey, and critically evaluate BPA contamination in meat products, including level of BPA and/or metabolites present, exposure route, and recent advancements in the analytical procedures of these compounds from meat and meat products. The contribution of meat and meat products to the total dietary exposure of BPA ranges between 10 and 50% depending on the country and exposure scenario considered. From can lining materials of meat products, BPA migrates towards the solid phase resulting higher BPA concentration in solid phase than the liquid phase of the same can. The analytical procedure is comprised of meat sample pre-treatment, followed by cleaning with SPE, and chromatographic analysis. Considering several potential sources of BPA in industrial and home culinary practices, BPA can also accumulate in non-canned or raw meat products. Very few scientific studies have been conducted to identify the amount in raw meat products. Similarly, analysis of metabolites and identification of the origin of BPA contamination in meat products is still a challenge to overcome.

This study aimed to examine the chemical makeup and antioxidant properties of hydroalcoholic extracts obtained from Lonicera japonica Thunb. Thunb. (honeysuckle) flowers collected from two geographically distinct regions of Romania: a mountainous region (Făgăraș Mountainous, Brașov) and a coastal region (Black Sea, Constanța). Our research revealed that the extracts from these two regions differed significantly in terms of their total polyphenol, flavone, and phenolic carboxylic acid content, as well as their antioxidant activity. These results suggest that climatic factors play a crucial role in shaping the phytochemical profile and antioxidant activity of Lonicera japonica Thunb. Thunb. extracts. Therefore, these findings could potentially contribute to the development of standardized herbal preparations and nutraceutical products.

The Drinking Water Directive (EU) 2020/2184 includes the parameter microcystin-LR, a cyanotoxin, which drinking water producers need to analyze if the water source has potential for cyanobacterial blooms. In light of the increasing occurrences of cyanobacterial blooms worldwide and given that more than 50 percent of the drinking water in Sweden is produced from surface water, both fresh and brackish, the need for improved knowledge about cyanotoxin occurrence and cyanobacterial diversity has increased. In this study a total of 98 cyanobacterial blooms were sampled in 2016-2017 and identified regarding their toxin production and taxonomical compositions. The surface water samples from freshwater lakes throughout Sweden including brackish water from 8 east coast locations along the Baltic Sea were analyzed for their toxin content with LC-MS/MS and taxonomic composition with 16S rRNA amplicon sequencing. Both the extracellular and the total toxin content was analyzed. Microcystin´s prevalence was highest with presence in 82% of blooms, of which as free toxins in 39% of blooms. Saxitoxins were found in 36% of blooms in which the congener decarbamoylsaxitoxin (dcSTX) was detected for the first time in Swedish surface waters at four sampling sites. Anatoxins were most rarely detected, followed by cylindrospermopsin, which were found in 6% and 10% of samples, respectively. As expected, nodularin was detected in samples collected from the Baltic Sea only. The cyanobacterial operational taxonomic unit (OTU) with the highest abundance and prevalence could be annotated to Aphanizomenon NIES-81 and the second most profuse cyanobacterial taxon to Microcystis PCC-7914. In addition, two correlations were found, one between Aphanizomenon NIES-81 and saxitoxins and another between Microcystis PCC-7914 and microcystins. This study is of value to drinking water management and scientists involved in recognizing and controlling toxic cyanobacteria blooms.

This study presents an innovative analytical method for the detection and quantification of 14 β-agonists in pork using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method involves the use of a solid phase microextraction (SPME) probe, consisting of a wooden tip coated with polyacrylonitrile (PAN) and molecularly imprinted polymer (MIP). This probe has been integrated into an automated extraction device, designed in our laboratory to extract the target analytes prior to LC-MS/MS analysis. Compared to the traditional solid-phase extraction (SPE) procedure, the proposed approach significantly reduces the costs related to adsorbent ma-terial and solvent consumption, and shortens purification time, thereby reducing both the tech-nical workload and associated expenses. The methodology has been carefully validated in ac-cordance with the National Standard of the People's Republic of China (GB/T 22286-2008), evalu-ating key parameters including the limit of detection (LOD), limit of quantification (LOQ), matrix effects, linearity, and both intra-day and inter-day precision. The average recovery rates for all β-agonists were observed to lie between 71.6% and 82.2%, with relative standard deviations (RSDs) consistently below 15% for both types of precision. The LOD and LOQ values were found to range from 0.09 to 0.39 μg/kg and 0.27 to 0.99 μg/kg, respectively. In terms of accurate detection of positive samples, the developed method demonstrated a closer approximation to the true value of the sample compared to the National Standard method GB/T 31658.22-2022. Overall, the proposed method exhibits substantial promise for the routine assessment of β-agonists in pork and presents a practical alternative for compliance monitoring in regulatory settings.

This article summarizes studies using the AIQS-DB method to analyze various types of samples for different purposes. This is a method developed to simultaneously analyze nearly 1500 compounds and is widely used worldwide. The method is highly effective for environmental samples such as water, soil, sediment, and air. Recognizing its potential, this article aims to promote the further application and development of AIQS-DB in research related to food analysis and source tracing. Furthermore, if a suitable dataset can be constructed, it could help research quickly and cost-effectively discover the new bioactive compounds from plant medicine.

Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy, verified by bio-statistical analysis (Pearson correlation coefficient, R=0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

Metabolomic analysis of biological fluids and tissues has become an increasingly routine tool in the biological toolbox. However, challenges remain to be overcome, including developing strategies to maximise coverage of the metabolome without requiring large sample volumes. Here we describe a multimodal strategy that combines data using both LC-MS and GC-MS from a unique vial with a sample of plasma (20µl) or a sample of brain tissue (3mg). Using a split phase extraction the non-aqueous phase was analyzed by reversed phase (RP) LC-MS, whilst the aqueous phase was analyzed using hydrophilic liquid interaction chromatography (HILIC)LC-MS, with both phases also analysed using GC-MS after derivatization of the extract. Analytical performance was assessed in 7 rat cerebellum samples and a pilot study of 40 plasma samples (20 vs. 20: AD vs. healthy controls). The method, which uses four hours of instrument time, measured 20,707 metabolite features in brain samples and 17,266 in plasma samples, from those 44.1% features displayed CV’s below 15% and 75.2% below 30%. The method has potential to resolve subtle biological differences and to correlate metabolite composition directly to clinical outcomes including MMSE, age and ADCS-ADL. This method can acquire in the order of 20K metabolic features when low volumes are available.

The 5-hydroxymethylfurfural (HMF) is a six-carbon heterocyclic organic compound with two substituents an aldehyde group and an alcohol group. It is formed from saccharides as a result of two reactions: the Maillard reaction and the caramelization of saccharides. It has both a negative and a positive effect on the human body, which makes it, even more, an attractive research object. The objective of the study was to determine HMF content in honey samples available on the Polish market using three varied methods and compare the results obtained. The methodology used included: two spectrophotometric methods (by White and Winkler) and the HMF determination method based on the LC-MS/MS technique developed uniquely for this study. The determined HMF content in all tested honey samples did not exceed the applicable standard. All results are within the scope of the European Council Directive 2001/110/EC of 20 December 2001, i.e. they do not exceed HMF content in honey, which is 40 mg/kg.

This study is aimed to point out the possible application of the Enzyme-Linked ImmunoSorbent Assay (ELISA) test on table olives. Because olives, like many other foods, are not free from mycotoxin contamination, the main focus of this work was assessing the presence or absence of aflatoxins and ochratoxin A. The work was carried out in two phases. In the first phase, tests were carried out on commercial olives to assess the efficacy of the method and the kit (Veratox® 'Neogen' and AgraQuant® 'Romer Labs') by comparing them with the Liquid Chromatography coupled to a mass-mass spectrometer (LC-MS/MS); in order to understand if there were any interferences in antigen-antibody recognition; subsequently, mycotoxins were inoculated on the same samples with a known quantity, in order to assess the concentrations obtained and compare them to the expected ones. The second step involved modifying the method, so to improve the recovery of inoculated mycotoxins in the samples under study. Afterwards we evaluated the presence of mycotoxins in table olives supplied in the project. The results showed the effectiveness of the ELISA test, especially in the mycotoxin recovery phase. Indeed, after the modification of the mycotoxin extraction method, recoveries increased from 55% to over 70% for aflatoxins, and from 40% to over 55% for ochratoxin A. When analysing the samples provided by the above-mentioned company, the results showed some false positives, in particular in relation to ochratoxin A ’s analysis, mainly related to the first sample submission. Taking this into account, the ELISA test can be considered as an initial screening method applicable as a control programme for the evaluation of mycotoxins in olives which helps by saving time and costs. It should always be kept in mind that, if positive values should emerge, the LC-MS/MS analysis should be carried out.

Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. is abundant of vitamin C and bioactive compounds with high antioxidant activities. In this study, eight wild A. arguta accessions from different areas in Northeast China were collected. Some bioactive compounds were examined on the different tissues of different germplasms including four kinds of leaves, petioles and fruits. The method of UPLC-MS was used to detect the flavonoid compounds. The results showed that some bioactive compounds including vitamin C, soluble sugar, free amino acid, total phenolics and flavonoids content showed significant differences between six tissues of A. arguta accessions and showed significant variability with maturity. In eight accessions, the highest vitamin C content was found in young apical leaves of ‘CBS-6’ (7.47 mg/g fresh weight), and the highest soluble sugar content was in fruits of ‘CJ-1’ (196.52 mg/g fresh weight) and the highest total phenolic content and total flavonoids content were in young apical leaves of ‘CBS-11’ (3.48 mg/g fresh weight) and of ‘CBS-3’ (2.00 mg/g fresh weight), respectively. Ten flavonoid compounds including kaempferol, isorhamnetin and quercetin were detected in leaves, petioles and fruits. The total content of flavonoids were highest in young apical leaves (10219.84 µg·g-1) and the lowest in fruits (78.75 µg·g-1). Based on the comparison of the contents of several bioactive compounds, the two accessions ‘CJ-1’ and ‘CBS-8’ had relatively outstanding performance, and in the comprehensive evaluation of the antioxidant activity among different tissues, the young leaves had the strongest antioxidant activity. These results highlighted the antioxidant potentialities of A. arguta leaves as a major source of phenolics and vitamin C as well as flavonoids. It provided a theoretical basis for the utilization of leaves of A. arguta.

A novel, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the evaluation of OTS167 pharmacokinetics in mouse serum. SN-38 was optimized to be selected as the internal standard. Chromatographic separation was performed on a BDS Hypersil C18 column (100 x 2.1 mm, 5 µm) using gradient elution with mobile phase solvent A as water containing 0.1% formic acid and solvent B as acetonitrile containing 0.1% formic acid. The analysis was carried out using multiple reaction monitoring (MRM) with a triple-quadrupole mass spectrometer operated in the positive electrospray ionization mode. Mass transitions of 487.2 > 348.0 and 393.2 > 349.2 were monitored for OTS167 and SN-38 respectively. The standard calibration curve demonstrated high linearity at a range of 5-1000 ng/mL, with a coefficient of determination (r2) ≥ 0.996. The accuracy for OTS167 ranged from 92.3% to 102.2% and the precision was ≤ 12.7%. Recovery was consistent at about 83% to 89%. No significant matrix effect was observed. This method was successfully applied to monitor the pharmacokinetic profiles in mice over 24 h after ingestion of 5 mg/kg of OTS167. Maximum serum concentrations (3197 ng/mL) of OTS167 was observed at 1.67 hour after OTS167 ingestion via garage at 5 mg/kg and area under the curve (AUC) was 28579 h*ng/mL, suggesting that OTS167 can be quickly absorbed after oral administration.

Bioprospecting native Australian plants offers potential discovery of latent and novel bioactive compounds. Promising cytotoxic and antibacterial activity of methanolic extracts of Pittosporum angustifolium and Terminalia ferdinandiana led to further fractionation and isolation using our laboratory’s bioassay guided fractionation protocol. Hence, the aim of this study was to further evaluate the bioactivity of the fractions and subfractions and characterize bioactive compounds using liquid chromatography mass spectroscopy (LC-MS/MS) and gas chromatography MS (GC-MS). Compounds tentatively identified in P. angustifolium Fraction 1 using LC-ESI-QTOF-MS/MS were chlorogenic acid and/or neochlorogenic acid, bergapten, berberine, 8’-epitanegool and rosmarinic acid. GC-MS analysis data showed the presence of around 100 compounds, mainly comprising of carboxylic acids, sugars, sugar alcohols, amino acids, and monoalkylglycerols. Furthermore, the fractions obtained from T. ferdinandiana flesh extracts showed no cytotoxicity, except against HT29 cell lines, and only Fraction 2 exhibited some antibacterial activity. The reduced bioactivity observed in the T. ferdinandiana fractions could be attributed to the potential loss of synergy as compounds become separated within the fractions. As a result, further fractionation, and separation of compounds in these samples were not pursued. However, additional dose-dependent studies are warranted to validate the bioactivity of T. ferdinandiana flesh fractions, particularly since this is an understudied species. Moreover, LC-MS/GC-MS studies confirm the presence of bioactive compounds in P. angustifolium Fraction 1/subfractions which helps to explain the significant acute anti-cancer activity of this plant. The screening process designed in this study has the potential to pave the way for developing scientifically validated phytochemical/bioactivity information on ethnomedicinal plants, thereby facilitating further bioprospecting efforts, and supporting the discovery of novel drugs in modern medicine.

Cholesterol-derived bile acids (BAs) affect numerous physiological functions such as glucose homeostasis, lipid metabolism and absorption, intestinal inflammation and immunity, as well as intestinal microbiota diversity. Diet influences the composition of the BA pool. The present study analyzes the impact of a dietary supplementation with a freeze-dried blueberry powder (BBP) on the fecal BA pool composition. The diet of 11 men and 13 women at risk for metabolic syndrome was supplemented with 50g/day of BBP for 8 weeks, and feces were harvested before (pre) and after (post) BBP consumption. BAs were profiled using liquid chromatography coupled to tandem mass spectrometry. No significant changes in total BAs were detected when comparing pre- vs post-BBP consumption samples. However, post-BBP consumption samples exhibited significant accumulations of glycine-conjugated BAs (p=0.04), glycochenodeoxycholic (p=0.01) and glycoursodeoxycholic (p=0.01) acids, as well as a significant reduction (p=0.03) of the secondary BA levels, when compared to pre-BBP feces (p=0.03). In conclusion, the fecal bileacidome is significantly altered after the consumption of BBP for 8 weeks. While additional studies are needed to fully understand the underlying mechanisms and physiological implications of these changes, our data suggest that the consumption of blueberries can modulate toxic BAs elimination.

Synthetic cannabinoids are a series of synthetic substances that mimic the effects of natural cannabinoids and produce a much stronger toxicity than natural cannabinoids, which have become the most abused family of new psychoactive substances. A solid-phase extractive-liquid chromatography-triple quadrupole/linear ion trap mass spectrometry method was developed to determine 11 synthetic cannabinoids in rat urine. The factors affecting recovery were optimized, and Oasis HLB was selected to extract synthetic cannabinoids simultaneously. The results showed that the linear correlation coefficients of the synthetic cannabinoids ranged from 0.993 to 0.999, and the limit of quantitation ranged from 0.01 to 0.1 ng/mL, and the spiked recoveries ranged from 69.90% to 118.39%. This method has the advantages of good purification ability, simple operation, and good reproducibility, and can be used for the high sensitivity analysis of various synthetic cannabinoids in urine.

Acrylamide is probably carcinogenic to humans (International Agency for Research on Cancer, group 2A) with major occurrence in heated, mainly carbohydrate-rich foods. For roasted coffee, a European Union benchmark level of 400 µg/kg acrylamide is of importance. Regularly, the acrylamide contents are controlled using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). This reference method is reliable and precise but laborious because of the necessary sample clean-up procedure and instrument requirements. This research investigates the possibility of predicting the acrylamide content from proton nuclear magnetic resonance (NMR) spectra that are already recorded for other purposes of coffee control. In the NMR spectrum acrylamide is not directly quantifiable, so that the aim was to establish a correlation between the reference value and the corresponding NMR spectrum by means of a partial least squares (PLS) regression. Therefore, 40 commercially available coffee samples with already available LC-MS/MS data and NMR spectra were used as calibration data. To test the accuracy and robustness of the model and its limitations, 50 coffee samples with extreme roasting degrees and blends were additionally prepared as test set. The PLS model shows an applicability for the varieties C. arabica and C. canephora, which were medium to very dark roasted using drum or infrared roasters. The root mean square error of prediction (RMSEP) is 79 µg/kg acrylamide (n=32). The PLS model is judged as suitable to predict the acrylamide values of commercially available coffee samples. On the other hand, very light roasts containing more than 1000 µg/kg acrylamide are currently not suitable for PLS prediction.

Exposure to bisphenol analogues can occur in several ways throughout the food production chain with their presence at higher concentrations representing a risk to human health. This study aimed to develop effective analytical methods to simultaneously quantify BPA and fifteen bisphenol analogues (i.e., bisphenol AF, bisphenol AP, bisphenol B, bisphenol BP, bisphenol C, bisphenol E, bisphenol F, bisphenol G, bisphenol M, bisphenol P, bisphenol PH, bisphenol S, bisphenol Z, bisphenol TMC and tetramethyl bisphenol F) present in canned foods and beverages. Samples of foods and beverages available in the Swiss and EU markets (n = 22) including canned pineapples, ravioli and beer were prepared and analyzed using QuEChERS GC-MS. The quantification method was compared to a QuEChERS LC-MS/MS analysis. This allowed for the selective and efficient quantitative simultaneous analysis of bisphenol analogues. Quantities of these analogues were present in 20 of the 22 samples tested with the most frequent analytes at higher concentrations; BPA and BPS being discovered in 78% and 48% of cases respectively. The study demonstrates the robustness of QuEChERS GC-MS for determining low quantities of bisphenol analogues in canned foods. However, further studies are necessary to achieve full knowledge of the extent of bisphenol contamination in the food production chain and its associated toxicity.

An ellagitannin-derived metabolite Urolithin A (UA) has emerged as a potential therapeutic agent for metabolic disorders due to its antioxidant, anti-inflammatory, and mitochondrial function-improving properties, but its efficacy in protecting against ER stress remains underexplored. The endoplasmic reticulum (ER) is a cellular organelle involved in protein folding, lipid synthesis, and calcium regulation. Perturbations in these functions can lead to ER stress, which contributes to the development and progression of metabolic disorders such as metabolic-associated fatty liver disease (MAFLD). In this study, we identified a novel target protein of UA and elucidated its mechanism for alleviating palmitic acid (PA)-induced ER stress. CETSA-LC-MS/MS analysis revealed that UA binds directly to the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), an important regulator of calcium homeostasis in mitochondria-associated ER membranes (MAMs). As an agonist of SERCA, UA attenuates abnormal calcium fluctuations and ER stress in PA-treated liver cells, thereby contributing to cell survival. In addition, UA inhibits PA-induced mitochondrial ROS and lipid accumulation, further supporting its protective role. The lack of UA activity in SERCA knockdown cells suggests that UA regulates cellular homeostasis through its interaction with SERCA. Collectively, our results demonstrate that UA protects against PA-induced ER stress and enhances cell survival by regulating calcium homeostasis in MAM through SERCA. This study highlights the potential of UA as a therapeutic agent for metabolic disorders associated with ER stress.

Due to similarities in genetics, cellular response, and behavior, Drosophila is used as a model organism in addiction research. A well described behavioral response examined in flies is the induced increase in locomotor activity after a single dose of volatilized cocaine (vCOC) and volatilized methamphetamine (vMETH) known as sensitivity, and the escalation of the locomotor response after the repeated dose, known as the locomotor sensitization. However, knowledge about how vCOC and vMETH affect different neurotransmitter systems over time is scarce. We used LC-MS/MS to systematically examine changes in the concentration of neurotransmitters, metabolites and non-metabolized COC and METH in the whole head homogenates of male flies one to seven hours after single and double vCOC or vMETH administrations. vMETH leads to complex changes in the levels of examined substances over time, while vCOC strongly and briefly increases concentrations of dopamine, tyramine and octopamine followed by a delayed degradation into N-acetyl dopamine and N-acetyl tyramine. The first exposure to psychostimulants leads to significant and dynamic changes in the concentrations relative to the second administration when they are more stable over several hours. Further investigations are needed to understand neurochemical and molecular changes post-psychostimulant administration.

A newly UHPLC-MS/MS method development and validation for S-Allyl Cysteine was used to evaluate the comparative pharmacokinetic parameters. SC PLGA NPs were developed by the emulsion solvent evaporation method. SC PLGA NPs showed their drug loading and encapsulation efficiency i.e. 5.13±0.10% and 82.36±4.01%, respectively. SC PLGA NPs showed a spherical morphology on an average size (134.8±4.61nm), PDI: 0.277±0.004, and −25.3±1.03mV zeta-potential is suitable for oral delivery. Development and validation of the UHPLC-MS/MS bioanalytical method were performed successfully for PK-parameters examinations with 1.219-minutes RT, MS (162.00/73.10), and a total run-time was 2.0-minutes. 1.0–1000.0ng/mL was a linear-range with inter & intra-day accuracy (92.55–99.40%) followed by precision (1.88–4.23%). SC PLGA NPs oral bioavailability was significantly higher (**p<0.01) as compared to SC-S treated groups (iv & oral). We found that the antimicrobial activity of SC PLGA NPs was more effective than pure S-Allyl-L-Cysteine with significant results (p<0.01) as compared to SC-S. SC PLGA NPs showed fitted physicochemical and enhanced antimicrobial properties which can be helpful for oral administration. On the basis of our observations, SC PLGA NPs suggested the highest potential for the improvement of oral bioavailability with a sustained and controlled release of S-Allyl-L-Cysteine delivery.

Adenosine is a multifunctional nucleoside with several roles across various levels in organisms. Beyond its intracellular involvement in cellular metabolism, extracellular adenosine potently influences both physiological and pathological processes. In relation to its blood level, adenosine impacts the cardiovascular system, such as heart beating rate and vasodilation. To exploit the adenosine levels in the blood, we employed the liquid chromatography method coupled with mass spectrometry (LC-MS). Immediately after the collection, a blood sample mixed with acetonitrile solution that is either enriched with 13C-labeled adenosine or a newly generated mixture is transferred into the tubes containing the defined amount of 13C-labeled adenosine. The 13C-enriched isotopic adenosine is used as an internal standard, allowing for more accurate quantification of adenosine. This novel protocol for LC-MS-based estimation of adenosine delivers a rapid, highly sensitive, and reproducible means for quantitative estimation of total adenosine in blood. The method also allows quantification of a few catabolites of adenosine, i.e., inosine, hypoxanthine, and xanthine. This advancement provides the analytical tool that has the potential to enhance our understanding of adenosine's systemic impact and pave the way for further investigations into its intricate regulatory mechanisms.

The extraction method for simultaneously analyzing fatty acids (Myristic acid, cis-Palmitvaccenic acid, Palmitic acid, Palmitic-9,9-d2 acid, Oleic acid, Stearic acid and Stearic-2,2-d2 acid) related to biodiesel fuel in large algae was developed. The introduction of trimethylaminoethyl (TMAE) derivatives into the hydroxyl group (-OH) functional group of the analyte was able to improve ionization and obtain high sensitivity. Liquid Chromatography (LC) was used to investigate the optimal mobile phase composition and flow rate for the separation of five derived fatty acids. In this study, a solid phase extraction (SPE) column was introduced for efficient extraction of fatty acids, and the optimal conditions for SPE were set by comparing the recovery rate according to the solvent for each step (loading, washing, and elution) of SPE from macroalgae.

Anti-microbial drugs and coccidiostat compounds are commonly used in poultry farming. These compounds are subsequently excreted and released into the environment via broiler litter (BL) and can re-enter the food chain as fertilizer and/or animal feed. Residues of such drugs can encourage the appearance of antibiotic-resistant bacteria (ARB), as well as toxicity, when found in animal feed. The aim of this study was to develop a multi-residue analytic method for assessing 30 an-ti-microbial drugs and coccidiostats associated with BL and investigate the effects of stacking treatment on anti-microbial and coccidiostat residue degradation in BL used for ruminant feed at the lab-scale. Today, most analytical methods used to identify and quantitate these drug residues are traditional, and may be specific to some antimicrobials. All call for lengthy sample preparation and may present limitations in assessing broiler litter. Such conventional mul-ti-anti-microbial/coccidiostat residue detection assays employ liquid-liquid extraction (LLE) and solid phase extraction (SPE) for sample purification. In our research, LLE and SPE were replaced by Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) clean-up steps for 30 an-ti-microbials and coccidiostats used in broiler farming and detection by liquid chromatography mass spectrometry (LC/MS/ MS). Using this method, recoveries were 70-120%, with the exception of norfloxacin and narasin, which showed recoveries of 67 and 68%, respectively. This analytical meth-od results show that eight samples were not detected in 42 samples, while in 34 samples, one or more drugs were present. Eighteen anti-microbial and coccidiostat residues were detected dur-ing one year sample collaction. The most frequently detected residues were narasin, nicarbazin, monensin, robenidine, oxytetracycline, chlortetracycline, norfloxacin and sulfachloropyridazine, seen at levels from 0.4 to 8.9 mg•kg-1 (ppm). Nineteen anti-microbials and one coccidiostat were degraded to 95% by the stacking treatment. However, tilmicosin and eight coccidiostats persisted in litter (26-100%). The use of antimicrobials and coccidiostats in animal farming is of paramount concern with respect to human, animal and environmental contamination issues and as a source of resistant pathogenic bacteria. Still, most farmers use BL as ruminant feed and fertilizer without processing. Even though the effects of the stacking treatments on degradation of antimicrobials were significantly high, the degradation of coccidiostats and tilmicosin was limited. Clearly, BL should be treated and supervised before use as feed supplement or for land application. Hence, the present method, designed to support these goals, was optimized in terms of sensitivity and se-lectivity for the analytes addressed.

A comprehensive lipid profile was analyzed in patients with non-small cell lung cancer (NSCLC) using nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. The study investigated 297, 202, and 166 lipids in saliva, plasma, and fecal samples, respectively, comparing NSCLC patients to healthy controls. Lipids with significant changes (&gt;2-fold, p&lt;0.05) were further analyzed in each sample type. Both saliva and plasma exhibited similar lipid alteration patterns in NSCLC, but saliva showed more pronounced changes and fecal lipids had weak correlation with those of saliva and plasma. Total triglycerides (TGs) increased (&gt;2–3 folds) in plasma and saliva but decreased in fecal samples. Three specific TGs (50:2, 52:5, and 54:6) were significantly increased in NSCLC across all sample types. A common ceramide species (d18:1/24:0) decreased in both plasma and saliva but increased in fecal samples. Additionally, phosphatidylinositol 38:4 decreased by approximately 2-fold in plasma and saliva. Phosphatidylserine 36:1 was selectively detected in saliva and showed a subsequent decrease, making it a potential biomarker for predicting lung cancer. The study identifies 27 salivary, 10 plasma, and 16 fecal lipids as candidate markers for NSCLC by statistical evaluations. Moreover, it highlights the potential of saliva in understanding changes in lipid metabolism associated with NSCLC.

Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P was linked to the pathological outcome with inflammation, cancer metastasis or angiogenesis etc. In this regard, the SPHK/S1P axis regulation has been a specific issue in anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhance the serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs which eventually induced an unusual environment of the high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase) that contributes a partial increase on Cers. Collectively, a dansyl-modified DPF-543 relatively en-hanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.

The aquaculture industry has become a sustainable provider of food to humans. Remaining challenges include disease issues, as well as ethical concerns for discomfort and stress among farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid-liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g and the limit of quantification was 0.34 ng/g, and the method was linear (R2>0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a CV of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

Tear fluid is emerging as a valuable resource for biomarker discovery, but its low sample volume and dynamic composition pose significant challenges for analytical testing. Most tear proteomics studies have focused on samples collected with Schirmer strips, while capillary tube collection has received less attention. To address these challenges, we developed a novel in-capillary trypsin digestion workflow that requires as little as 0.5 μL of tear fluid for bottom-up shotgun proteomics. This method uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify a higher number of proteins with enhanced efficiency compared to previously reported methods, which typically required 5-10 μL of pooled tear fluid. Using the same microcentrifuge tube for both tear collection and sample processing, this streamlined workflow simplifies sample handling and minimizes both sample loss and experimental errors associated with sample transfer. This method also efficiently reduced sample processing time to under 2 hours prior to overnight trypsin digestion, compared to the 5-8 hours required by other methods. With this workflow, we identified 500-800 proteins per 0.5 μL sample without the need for fractionation, allowing for at least three technical replicates. This optimized workflow greatly enhances the efficiency of tear proteomics for biomarker discovery.

Brazil chicken production is around 13 million tons and about a third is exported to over 150 countries, placing Brazil as the world largest chicken meat producer, and therefore it is crucial to follow the legislation of all importer markets. This study conducted a survey by chance in 45 meat industries able to export. Therefore, 2580 chicken meat samples were collected and submitted to 11 analyte extraction and chromatographic verification of compliance in an accredited laboratory. Ten chemical residues (amoxicillin, bacitracin, colistin, dinitolmide + zoalene, spectinomycin, roxarsone, tiamulin, tylosin, trenbolone acetate and virginiamycin) were investigated in chicken meat and one (halofuginone hydrobromide) in chicken liver. The results showed that no compound exceeded the maximum residue limits established by seven legislations. All residue concentrations found were below the method quantification limit, thereby confirming the capability of Brazilian chicken meat industries in complying to foreign markets.

Tree peony flowers are traditional ornamental and medicinal materials in China. In this study, 23 tree peony flowers at a broad color spectrum were analyzed. Gas chromatography-mass spectrometer (GC-MS) revealed that tree peony flowers are rich in sugars and organic acids. Up to 18 amino acids were identified by liquid chromatography-mass spectrometer (LC-MS), including all essential amino acids, except for methionine. The majority of amino acids were significant positively correlated with each other and were significant negatively correlated with glucose, fructose and galactose. A total of 11 polyphenols were identified in these tree fresh peony flowers by LC-MS. There was a high consistency in grouping peony flowers by using sugars and organic acids and amino acids, which differed from that based on color components and polyphenols. Tree peony flowers are also rich in K, Ca, Mg and Fe. In together, peony flowers can be a good resource of health-promoting compounds.

Asparagus shoots are worldwide consumed, although most of such species have a restricted range, and several taxa still remain unstudied. In this work, a total of four taxa from different locations were scrutinized and compared with cultivated A. officinalis. All shoots were screened for saponins by LC-MS, and in vitro antiproliferative activities against the HT-29 colorectal cancer cell line (by MTT assay). The total saponins (TS) contained in the crude extracts ranged from 710.0 (A. officinalis) to 1258.6 mg/100 g dw (A. acutifolius). It stands out the richness of compounds detected in this work: a total of 47 saponins have been detected and quantified in the edible parts (shoots) of 5 taxa of Asparagus. The structure of all the saponins found present skeletons of the furostane and spirostane type. In turn, the structures with a furostane skeleton are divided into unsaturated and dioxygenated, both in the 20-22 position. The sum of dioscin and derivatives varied largely along the studied taxa. It reached the following percentages of TS: 27.11 (A. officinalis), 18.96 (A. aphyllus), 5.37 (A. acutifolius), and 0.59 (A. albus), while in A. horridus such compound remains undetected. Aspachiosde A, D, and M varied largely among samples, while a total of seven aspaspirostanosides were characterized in the analyzed species. Hierarchical cluster analysis of the saponin profiles clearly separated the various taxa and demonstrated that the taxonomic position is more important than the place from which the samples were acquired. Thus, saponin profiles have chemotaxonomic significance in Asparagus taxa. The MTT assay showed dose- and time-dependent inhibitory effects of several saponins extracts on HT-29 cancer cells, standing out the cell growth inhibition exercised by A. albus and A. acutifolius (GI50 of 125 and 175 µg/mL). This work constitutes a whole approach to evaluate the saponins from shoots of different Asparagus taxa and provide arguments for use them as functional foods.

Macrophages are the key effector cells of innate immunity which show two polarized states: M1, classically activated and M2, alternatively activated. Tumor-associated macrophages (TAMs) which usually show M2 polarization, are immunosuppressive cells that enhance tumor metastasis and invasion. Also, enrichment of TAMs is known to be closely associated with poor prognosis of cancer patients. Therefore, TAMs are considered to be promising targets for immunotherapy. Importantly, tumor-derived exosomes emerged as a crucial player in immune regulation which can remodel the tumor microenvironment towards immunosuppressive state. Of note, few studies have shown that exosomes could induce polarization of macrophages towards M2 type in tumor condition, while others showed activation of M1 or mixed phenotype. Considering the role of TAMs in cancer, there is an urgent need to decipher the exosome-mediated cross-talk between tumor cells and macrophages.For this, we used murine model of Dalton’s lymphoma (DL), wherein, firstly, we evaluated increased exosome burden by assessing exosome level in serum as well as in various tissues of tumor-bearing host. Next, proteomic profiling of lymphoma-derived exosomes was done which revealed the presence of immunomodulatory proteins. Of note, these proteins were known to alter the macrophage polarization. In order to assess the effect of these exosomes on macrophages, we performed in vitro study using RAW264.7 cells. In vitro study revealed that frequent uptake of exosomes mediated a morphological change in macrophages which reduced their phagocytic activity. In parallel, exosomes increased reactive oxygen species (ROS) level and inhibited LPS-induced nitric oxide (NO) level in macrophages. Also, exosomes upregulated the expression of arginase-1 (an M2 marker) in macrophages but decreased the LPS-induced nitric oxide synthase 2 (NOS2) (an M1 marker) expression. Moreover, we observed a pro-tumoral cytokine profile in macrophages incubated with exosomes. Our study suggests that exosome load increases in lymphoma-bearing hosts systemically. Importantly, lymphoma-derived exosomes mediate the activation of macrophages towards pro-tumoral M2 type. Our results give an insight into the exosome-mediated tumor and immune cell cross-talk which could serve as an important prospect for targeting in cancer immunotherapy.

Mulberry is an economically significant crop for the sericulture industry worldwide. Stresses such as drought exposure have a significant influence on plant survival. Metabolome directly reflects plant physiological status; thus, a way to assess this impact is to perform a global metabolomic analysis. This study investigated the effect of drought stress on mulberry Yu-711 metabolic balance using a liquid chromatography-mass spectrometry (LC-MS) based on an untargeted metabolomic approach. For this objective, Yu-711 leaves were subjected to two weeks of drought stress treatment and control without drought stress. Multivariate and univariate statistical analyses highlighted numerous differentially-accumulated metabolic elements as a function of time and treatment. Drought stress led to a more differentiated metabolites response than the control. We found that the levels of total lipids and galactolipids, and phospholipids (PC, PA, PE) were significantly altered, producing 48% of the total differentially expressed metabolites. Fatty acyls were the most abundant lipids expressed and decreased considerably by 73.6%. Prenol lipids class of lipids increased in drought leaves. Other classes of metabolites, including polyphenols( flavonoids and cinnamic acid), organic acid (amino acids), carbohydrates, benzenoids, and organoheterocyclic, all had a dynamic trend in response to the drought stress. However, their levels under drought stress generally decreased significantly compared to the control. These results provide an overview of the metabolic profile of the mulberry plant through differentially-accumulated compounds and provide a better understanding of global plant metabolic changes in defense mechanisms.

Objective: To study the changes in urine metabolism in female water polo players before and after high-intensity training by using ultra-high performance liquid chromatography-mass spectrometry, and to explore the biometabolic characteristics of urine after training and competition. Methods: Twelve young female water polo players (except goalkeepers) from Shanxi Province were selected. A 4-week formal training was started after one week of acclimatization according to experimental requirements. Urine samples (5 ml) were collected before formal training, early morning after 4 weeks of training, and immediately after 4 weeks of training matches, and labeled as T1, T2, and T3, respectively. The samples were tested by LC-MS after pre-treatment. XCMS, SIMCA-P 14.1, and SPSS16.0 were used to process the data and identify differential metabolites. Results: On comparing the immediate post-competition period with the pre-training period (T3 vs T1), 24 differential metabolites involved in 16 metabolic pathways were identified, among which niacin and niacinamide metabolism and purine metabolism were potential post-competition urinary metabolic pathways in the untrained state of the athletes. On comparing the immediate post-competition period with the post-training period (T3 vs T2), 10 metabolites involved in 3 metabolic pathways were identified, among which niacin and niacinamide metabolism was a potential target urinary metabolic pathway for the athletes after training. Niacinamide, 1-methylnicotinamide, 2-pyridone, L-Gln, AMP, and Hx were involved in two metabolic pathways before and after the training. Conclusion: Differential changes in urine after water polo games are due changes in the metabolic pathways of niacin and niacinamide.

(1) Background: Food and Feed Safety Legislation does not concern all the mycotoxins generated by Penicillium and Aspergillus spp. Certain mycotoxins, including cyclopiazonic acid (CPA), penicillic acid (PA), roquefortine C (ROQ C), mycophenolic acid (MPA), and gliotoxin regarded as having lower toxicity levels, hence not included in food and feed legislation; (2) Methods: Presence of these substances in maize and wheat grown in Albania across two consecutive harvesting seasons was investigated by liquid chromatography and mass spectrometry (LC-MS/MS); (3) Results: The findings indicated the presence of these mycotoxins in maize grain but not in wheat grain. In the 2014 season, they exhibited a higher contamination incidence than the 2015 season. The most commonly detected Mycotoxin was MPA, followed by CPA and ROC toxin, while Penicillium acid and gliotoxin were detected. The MPA revealed a concentration range of 72.92–3447.25 μg/kg, with a mean value of 1063.89 μg/kg. Mycophenolic acid was detected in the maize samples collected during the 2015 season. (4) Conclusions: These findings suggest that focusing the investigation only on 'controlled' mycotoxins will not produce a proper risk assessment and may not adequately address the possible harmful impacts of mycotoxins on human and animal health due to mycotoxins co-occurrence.

The spread of SARS-CoV-2 variants of concern (VOCs), is of great importance to the whole community, since their genetic changes may increase transmissibility, disease severity and reduce effectiveness of vaccines. Moreover, these changes may lead to failure of diagnostic measures, thus variant-specific diagnostic methods are essential. To date, genetic sequencing is the gold standard method to discriminate between variants, however it is time consuming (several days) and expensive. Therefore, the development of rapid diagnostic methods for SARS-CoV-2 in accordance with its genetic modification is of great importance. In this study, we introduce a Mass-Spectrometry (MS)-based methodology for the diagnosis of SARS-CoV-2 in clinical specimens, a methodology which enables universal identification, alongside with variant-specific discrimination. The universal identification of SARS-CoV-2 is based on conserved markers shared by all variants, while the identification of the specific variant relies on variant-specific markers. Determining a specific set of peptides for a given variant consists of a multistep procedure, starting with in-silico search for variant-specific tryptic peptides, followed by tryptic digest of a cell-cultured SARS-CoV-2 variant and identification of these markers by HR-LC-MS/MS analysis. As a proof of concept, this approach was demonstrated for four representative VOCs, in comparison with the wild-type Wuhan reference strain. For each variant, at least two unique markers, derived mainly from the spike (S) and nucleocapsid (N) viral proteins, were identified. This methodology is specific, rapid, easy to perform and inexpensive, therefore can be applied as a general diagnostic tool of pathogenic variants.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC– MS/MS) method has been developed for the simultaneous determination of atorvastatin (ATOR), ezetimibe (EZM) and their three metabolites including o-hydroxyl atorvastatin (o-OH ATOR), p-hydroxyl atorvastatin (p-OH ATOR), and ezetimibe-glucuronide (EZM-G) in human plasma using benzyl paraben (BP) as internal standard (IS). The analytes and IS were ionized using ESI positive ion mode (ATOR, o-OH ATOR, and p-OH ATOR), ESI negative ion mode (EZM, EZM-G, and BP), and operated in multiple reaction monitoring (MRM) mode. They were next extracted by salting-out assisted liquid–liquid extraction with acetonitrile, and then analyzed by liquid chromatography on a reversed-phase chromatographic column (50 mm × 4.6 mm; 3.5 µm), using a mixture of ac-etonitrile and an acetic acid solution (0.5%) as the mobile phase, showing high extraction effi-ciency (>70%), and a minimized matrix effect. The method was satisfactorily validated, and showed excellent linearity over wide concentration ranges of 0.06–15 ng/mL, 0.6–150 ng/mL, 0.4–100 ng/mL, 0.12–30 ng/mL, and 0.05–3 ng/mL for EZM, EZM-G, ATOR, o-OH ATOR, and p-OH ATOR, respectively.

Nitazenes represent a New Synthetic Opioids sub-class, belonging to the New Psychoactive Substances (NPS). Their high pharmacological potency has led to numerous intoxications and fatalities, even at minimum doses. The aim of this study was to assess the stability of four nitazenes (etazene, flunitazene, isotonitazene and protonitazene) in dried blood spot (DBS) samples at different storage temperatures (room temperature and 4°C), using a newly developed and validated liquid chromatography-high resolution mass spectrometry method. Two concentrations, 1 and 5 ng/mL were chosen based on the available data on nitazene-related intoxications and stability was evaluated at day 0 (control), 1, 7 and 30. Results showed that all analytes at 1 ng/mL were not detectable after 30 days at room temperature; a similar pattern was observed for 1 ng/mL etazene and isotonitazene samples when stored at 4°C, whereas flunitazene and protonitazene decreased to a mean of 66% and 69% initial concentration, respectively, at day 30. Differently, all analytes at 5 ng/mL were quantified above 44% and 41% initial concentration at room temperature and 4°C, respectively, showing a higher stability. The study of nitazenes stability in DBS represents an important tool to determine the optimal samples storage conditions, such as temperature and time between sample collection and analysis.

Epirubicin, olaparib and ribociclib are three anti-tumoral molecules currently used in cancer therapy. Since serum levels of these drugs reveal a large inter-individual variability, validation of a fast, simple and reliable analytical method for quantitative determination of these molecules in human serum, with Therapeutic Drug Monitoring (TDM) purposes would be necessary. Papers in literature describe separation, identification and quantitation of the drugs in pharmaceutical formulas and biological matrices. The large majority of these approaches use protein precipitation for sample pre-treatment, followed by LC separation and tandem-mass-spectrometry (MS/MS) detection. We propose an alternative method using Solid Phase Extraction (SPE) with Oasis PRiME HLB® cartridges followed by high-performance liquid chromatography (HPLC) using a C18 (4.6 × 50 mm) column. A gradient mobile phase with 0.1% formic acid/acetonitrile was utilized. MS detection in single ion recording (SIR) mode was employed. A 13-minutes run-time analysis, including column re-equilibration was assessed. Data for all molecules were validated according to ICH Guidelines. Calibration curves for all analytes were linear with correlation coefficient larger than 0.997. Values for precision were less than 6%. The method was applied on serum samples from patients up-taking the drugs, proving its suitability in TDM assisted adjustment of doses in therapy.

Glioblastoma is a highly malignant brain tumor consisting of heterogeneous cellular population. The transformed metabolism of glioblastoma cells supports their growth and division on the background of their milieu. One might hypothesize that the transformed metabolism of a primary glioblastoma could be well adapted to limitations in the variety and number of substrates imported into the brain parenchyma and present it their microenvironment. Additionally, the phenotypic heterogeneity of cancer cells could promote the variations among their metabolic capabilities regarding the utilization of available substrates and release of metabolic intermediates. With the aim to identify the putative metabolic footprint of different types of glioblastoma cells, we exploited the possibility for separation of polar and ionic molecules present in culture media or cell lysates by hydrophilic interaction liquid chromatography (HILIC). The mass spectrometry (MS) was then used to identify and quantify the eluted compounds. The introduced method allows the detection and quantification of more than 150 polar and ionic metabolites in single run, those may be present either in culture media or cell lysates and provide data for polaromic studies within metabolomics. The method was applied to analyze the culture media and cell lysates derived from two types of glioblastoma cells, T98G and U118. The analysis revealed that even the both types of glioblastoma cells share several common metabolic aspects, they also exhibit differences in their metabolic capability. This finding agrees with the hypothesis about metabolic heterogeneity of glioblastoma cells. Furthermore, the combination of both analytical methods, HILIC-MS, provides a valuable tool for metabolomic studies based on the simultaneous identification and quantification of a wide range of polar and ionic metabolites - polaromics.

The dynamic interplay of signaling networks in most major cellular processes is characterized by the orchestration of reversible protein phosphorylation. Consequently, analytic methods like quantitative phospho-peptidomics has been pushed forward from a highly specialized edge-technique to a powerful and versatile platform for comprehensively analyzing the phosphorylation profile of living organisms. Despite enormous progress in instrumentation and bioinformatics, a major problem remains a high number of missing values caused by the experimental procedure due to either a random phospho-peptide enrichment selectivity or borderline signal intensities, which both cause the exclusion for fragmentation using the commonly applied data dependent acquisition (DDA) mode. Consequently, an incomplete dataset reduces confidence in the subsequent statistical bioinformatic processing. Here, we successfully applied data independent acquisition (DIA) by using the filamentous fungus Magnaporthe oryzae as model organism and could prove that while maintaining data quality (such as phosphosite and peptide sequence confidence), the data completeness increases dramatically. Since the method presented here reduces the LC-MS/MS analysis from 3 h to 1 h and increases the number of phoshosites identified up to 10-fold in contrast to published studies in fungi, we pushed the phospho-proteomic technique beyond its current limits and could provide a sophisticated resource for investigation of signaling processes in filamentous fungi.

In recent years, growing attention has been directed towards the potential of plant extracts as great therapeutic agents in controlling oxidative stress and inflammation. Gymnema inodorum (Lour.) Decne, an indigenous plant locally grown in Northern Thailand, is known for its anti-diabetic activity. This study aims to explore the antioxidant properties and phenolic profile of column chromatographic fractionated G. inodorum leaf extract. The ethanolic-reflux extracts of G. inodorum leaves were subjected to column chromatography for the isolation of bioactive molecules. Total phenolic and flavonoid content were assessed using Folin–Ciocalteu and aluminium chloride colorimetric methods, respectively. Total antioxidant activity was evaluated by employing different antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic)acid (ABTS) scavenging assay and ferric reducing antioxidant power (FRAP) assay. High-Performance Liquid Chromatography (HPLC) with a C18 column was employed to determine the level of gymnemic acid, while Liquid Chromatography Quadrupole Time-of-Flight/Mass Spectrometry (LC-QTOF/MS) was used to identify potential phenolic compounds. The three separated fractions indicating A, B, and C, exhibited remarkable antioxidant activity, with total phenolic content of 0.294 ± 0.056, 0.085 ± 0.039, and 0.227 ± 0.041 mg QE/mL, and total flavonoid content of 0.274 ± 0.050, 0.309 ± 0.110, and 0.384 ± 0.007 mg QE/mL, respectively. Fraction A exhibited the highest DPPH and ABTS scavenging activity, while fraction C demonstrated significantly higher ferric-reducing power. HPLC analysis revealed a highest concentration of gymnemic acid in fraction C (0.393 ± 0.003 mg/g) compared to fraction A (0.159 ± 0.003 mg/g) and fraction B (0.142 ± 0.000 mg/g). The LC-QTOF/MS phenolic screening showed seven potential phenolic compounds in fraction A, seven compounds in fraction B, and four compounds in Fraction C. A significant relationship is found between antioxidant potential and total phenolic content, suggesting that phenolic compounds are the major contributors to the antioxidant activity of G. inodorum.

Jatropha multifida L. is a plant belong to Euphorbiaceae family used in Benin pharmacopoeia because of its medicinal properties. The objective of this work is to appreciate the therapeutic power of this plant through the evaluation of some of its biological activities. After the preliminary phytochemistry, the polyphenols and flavonoids were quantified and identified by the LC-MS-ESI. The antimicrobial power of the extracts was investigated by agar medium diffusion. The antioxidant power of the extracts was evaluated by the reduction of the DPPH radical, the ABTS radical cation, the feric ion (FRAP) and the lipid peroxidation (LPO). Anti-inflammatory activity was assessed by inhibition of protein (albumin) denaturation. Indeed, different phenolic and flavonoid compounds namely, 2-Hydroxybenzoic acid, o-Coumaroylquinic acid, Apigenin-apiosyl-glucoside, Luteolin-galactoside, Luteolin-glucoside, Luteolin-rhamnoside, Quercetin-glucoside, Quercetin-arabinoside, Dicaffeoyquinic acid, Kaempferol-rhamnoside were identify. The J. mutifida extracts has a bactericidal effect with reference strains of wich CMBs vary from 22.67 mg/ml (S. aureus, S. enteridis, L. monocytogenes and C. albicans) to 47.61 mg/ml (E. coli) and with the meat isolated strains (S. equorum, S. saprophiticus, S. haemoliticus, S. cohnii and S. lentus). Ethanolic extract show the highest DPPH radical scavenging activity (IC₅₀ = 0.72±0.03 mg/ml) while methanolic extract showed the highest ferric ion reduction (46.23±1.10 µgEAA/g). Contrary to the FRAP method, the reducing power with ABTS method of water-ethanolic extract was greater (0.49±0.11 mol ET/g). The same extract show the highest albumin denaturation inhibition power (97.31±0.35%) at 1000µg/ml. J. multifida extracts are rich in bioactive compound with good biological activity

Conventional fluorescent lamps used in tissue culture are costly light sources showing excessive wavelength emission-bandwidth that must be replaced by alternative, less costly and much lower power-consuming energy sources. The use of Light-Emitting Diodes (LEDs) is the best option due to their potential role as elicitors of secondary metabolite production in many plant models. Gynura procumbens(G. procumbens) is widely used for treating various diseases. Here, leaf explants were cultivated in MS medium supplemented with 0.5 mg/L of NAA and 2.0 mg/L of BAP for 30 days under white, blue, and red LEDs. Secondary metabolites were analyzed by HPLC and LC-MS. Blue LEDs elicited the highest antioxidant activity, total flavonoid, and phenolic content. Furthermore, the content of cyanidin-monoglucosides increased significantly increased under blue light.

Lemon balm extracts by dry plant material of Melissa officinalis were tested against their efficacy against for different fungal species Alternaria alternata, Fusarium oxysporum, Aspergillus flavus and Beauveria bassiana. The aqueous phase of the extract was evaluated for antioxidant, antifungal and also anti-aflatoxigenic activity. A synergism evaluation was also performed concerning, the small quantity of lemon balm polar essential oil compounds extracted into the infusion and the water soluble compounds in the extract. The experiments were conducted in solid cultures and the growth inhibition was demonstrated by measuring mycelial diameter. Additionally, the effect on conidia production was also demonstrated. Lemon balm was also used for in situ test on Pistachia vera seed against Aspergillus flavus growth and aflatoxin production. Results revealed enhancement of fungal growth by lemon balm extracts however total inhibition of aflatoxins production on Pistachia vera seeds was observed, and both actions were tried to attributed to volatile and water soluble compounds identified based on GC/MS, HPLC/DAD and LC/MS, and the observed antioxidant activity. Volatile and water - soluble compounds found to be in absolute synergism in mycelium growth enhancement and the observed anti-aflatoxigenic activity addressed to the high antioxidant activity observed and synergistic action between the other water soluble phenolic compounds identified in the extract. The findings of this study underline the high biological active potential of lemon balm extracts under various screening test since for the first time full phytochemical analysis of lemon balm extracts.

Epilepsy is a heterogenous neurological disorder characterized by recurrent unprovoked seizures, mitochondrial stress, and neurodegeneration. Hydrogen sulfide (H2S), a gasotransmitter, promotes mitochondrial function and biogenesis, elicits neuromodulation and neuroprotection, and may acutely suppress seizures. A major gap in knowledge remains in understanding the role of mitochondrial dysfunction and progressive changes in H2S levels following acute seizures and during epileptogenesis. We thus sought to quantify changes in H2S and its methylated metabolite (MeSH) via LC-MS/MS subsequent to acute maximal electroshock and 6 Hz 44 mA seizures in mice, as well as in the corneal kindled mouse model of chronic seizures. Plasma H2S was acutely reduced after a maximal electroshock seizure. H2S or MeSH levels in whole brain homogenate and expression of related genes in corneal kindled mice were not altered. However, plasma H2S and MeSH levels were significantly lower during kindling, but not after established kindling. Morever, we demonstrated a time-dependent increase in expression of mitochondrial membrane integrity-related proteins, Opa1, Mfn2, Drp1, and Mff during kindling, which did not correlate with gene expression. Taken together, short-term reductions in plasma H2S could be a novel biomarker for seizures. Future studies should further define the role of H2S and mitochondrial stress in epilepsy.

Seeds from several Ribes taxa were surveyed for phenolic compounds and in vitro antiproliferative activity against HT-29 colorectal cancer cells. Total phenolic compounds were analysed through the Folin-Ciocalteu procedure, while phenolic profiles were analysed by LC coupled to a single mass spectrometer Orbitrap using an electrospray interface (ESI). Antitumor effects were established using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Total phenolics ranged from 11.4 in R. alpinum to 94.8 mg of caffeic acid equivalents (CAE)/g in R. nigrum 'Koksa'. Concerning phenolic compounds, four were hydroxylated benzoic acids, four cinnamic acid derivatives, eight flavonoids, and nine flavonoid glycosides. The growth inhibition against HT-29 cancer cells was exercised much better by R. nigrum 'Koksa' and Ribes 'Erkeeni' (GI50 37 and 42 µg/ml). All Ribes extracts, except R. nigrum 'Hara katarlik', showed higher activity than that of R. rubrum (GI50 at 72 h: 99 µg/ml). Interestingly, the highly bioactive extract from Ribes 'Erkeeni' contains all detected phenolics, while R. nigrum 'Koksa' lacks only populnin. Therefore, the high bioactivity found for such extracts could be due to a synergy due to all detected compounds. This work constitutes a comprehensive action for expanding knowledge on the phenolic profiles and antitumor activity of GLA-rich Ribes seeds.

Chrysanthemum tea, a typical health tea with the same origin as medicine and food, is famous for its unique health benefits and flavor. To clarify the influence of metabolites of different varieties of chrysanthemum tea on the formation of taste and quality differences, nontargeted metabolomics combined with electronic tongue analysis were used to characterize the correlation between metabolites profiles and taste characteristics of different quality chrysanthemum tea. Thirteen metabolites were identified as the key metabolites of the sensory quality difference between Huangju and Jinsi Huangju tea. Kaempferol, luteolin, genistein, and some quinic acid derivatives were correlated with the ‘astringent’ and taste attributes. In contrast, l-(-)-3 phenyllactic acid and L-malic acid were found to be the ‘bitterness’ and ‘umami’ in chrysanthemum tea. KEGG pathway enrichment analysis showed that the flavonoid and flavonol biosynthesis pathway had important effects on the sensory quality of chrysanthemum tea.

Ferroptosis is a unique variety of non-apoptotic cell death, driven by massive lipid oxidation in an iron-dependent manner. Since Ferroptosis was introduced as a concept in 2012, it was shown it's essential role in the pathogenesis in neurodegenerative diseases and an important role in therapy-resistant cancer cells. Thus, a detailed molecular understanding of both canonical and alternative ferroptosis pathways are required. There is a set of widely used chemical agents to modulate ferroptosis using different pathway targets: Erastin blocks cystine-glutamate antiporter, system xc-; ML210 directly inactivate GPX4; L-buthionine sulfoximine (BSO) inhibits γ-glutamylcysteine synthetase, an essential enzyme for glutathione synthesis de novo. Most studies were focused on lipidomic profiling of model systems undergoing death in a ferroptotic modality.

The aim of the study was to gain deeper insights in the potential for polyclonal stimulation of PBMC from cows and pigs with banana lectin (BanLec) from Musa paradisiaca. BanLec induced a marked proliferative response in cow and pig PBMC. The response in pigs was even higher than to Concanavalin A. Molecular processes associated with respective responses were examined with differential proteome analyses. Discovery proteomic experiments was applied to BanLec stimulated PBMC and cellular and secretome responses were analyzed with label free LC-MS/MS. In PBMC, 3955 proteins were identified. After polyclonal stimulation with BanLec, 459 proteins showed significantly changed abundance in PBMC. In respective PBMC secretomes, 2867 proteins were identified with 231 differentially expressed candidates as reaction to BanLec stimulation. The transcription factor `E74 like ETS transcription factor 1 (ELF1)` was solely enriched in BanLec stimulated PBMC. BanLec induced secretion of several immune regulators, amongst them positive regulators of activated T cell proliferation and Jak-STAT signaling pathway. Top changed immune proteins were CD226, CD27, IFNG, IL18, IL2, CXCL10, LAT, ICOS, IL2RA, LAG3 and CD300C. BanLec stimulates PBMC of cows and pigs polyclonally and induces IL2 pathway and further proinflammatory cytokines. Proteomics data are available via ProteomeXchange with identifier PXD027505.

The breeding of stress-tolerant cultivated plants that would allow for a reduction in harvest losses and undesirable decrease in quality attributes requires a new quality of knowledge on molecular markers associated with relevant agronomic traits, on quantitative metabolic responses of plants on stress challenges, and on the mechanisms controlling the biosynthesis of these molecules. By combining metabolomics with genomics, transcriptomics and proteomics datasets a more comprehensive knowledge of the composition of crop plants used for food or animal feed is possible. In order to optimize crop trait developments, to enhance crop yields and quality, as well as to guarantee nutritional and health factors, that provides the possibility to create functional food or feedstuffs, the knowledge about the plants’ metabolome is crucial. Next to classical metabolomics studies, this review focusses on several metabolomics based working techniques, such as sensomics, lipidomics, hormonomics and phytometabolomics, which were used to characterize metabolome alterations during abiotic and biotic stress, to find resistant food crops with a preferred quality or at least to produce functional food crops are highlighted.

This study investigated metabolic changes associated with Bovine Leukemia Virus (BLV) infection in dairy cows, focusing on pre-parturition alterations. Of 145 cows examined, 42 (28.9%) were BLV-seropositive. 38% of infected cows showed high somatic cell counts indicative of subclinical mastitis, with 15 experiencing additional health issues such as ketosis, milk fever, and lameness. Despite these conditions, no significant differences in milk yield or composition were observed between infected and control groups. Metabolomic analysis conducted at -8 and -4 weeks prepartum revealed significant metabolic differences between BLV-infected and healthy cows. At -8 weeks, 30 serum metabolites were altered, including sphingomyelins, lysophosphatidylcholines, amino acids, and acylcarnitines, suggesting disruptions in membrane integrity, energy metabolism, and immune function indicative of early neoplastic transformations. By -4 weeks, the number of altered metabolites reduced to 17, continuing to reflect metabolic disruptions in cows with leukemia. Multivariate analysis highlighted distinct metabolic profiles between infected and control cows, identifying key discriminating metabolites such as choline, aspartic acid, phenylalanine, and arginine. Urine metabolomics revealed significant prepartum shifts in metabolites related to glucose, asymmetric dimethylarginine, and pyruvic acid, among others. The research confirmed metabolomics' efficacy in defining a BLV infection metabolic profile, elucidating leukosis-associated metabolic disruptions. This approach facilitates identification of BLV-infected cows and enhances understanding of infection pathophysiology, providing a foundation for advanced management and intervention strategies in dairy herds. The study underscores the profound impact of leukosis on metabolic processes and highlights urine metabolomics' utility in non-invasively detecting BLV infection, offering potential for improved herd health management.

Studies on the neurobiological causes of anxiety disorders suggest that the GABA system in-creases synaptic concentration and enhances the affinity of GABAA (type A) receptors for ben-zo-diazepine ligands. Flumazenil antagonizes the benzodiazepine binding site of the GABA (γ-aminobutyric acid) type /benzodiazepine receptor (BZR) complex in the central nervous sys-tem (CNS). The integration of the metabolites of flumazenil by LC-tandem mass spectrometry will complete understanding of the in vivo metabolism of flumazenil and accelerate radio-pharmaceutical inspection and registration. The main goal of our study was to investigate using reversed-phase HPLC (PR-HPLC) coupled with electrospray ionization triple quadrupole tan-dem mass spectrometry (ESI-QqQ MS) for the identification of flumazenil and its metabolites in a hepatic matrix. And a carrier-free nucleophilic fluorination with automatic synthesizer for [18F]flumazenil which applied to in vivo nano-positron emission tomography (Nano-PET)/computed tomography (CT) imaging and ex vivo bio-distribution used to analyze in normal rats. The study showed that 50% of the flumazenil was bio-transformed at 60 min by the rat liver homogenate, while one metabolite (M1) was a methyl transesterification product of flumazenil. In a rat liver microsomes system, two metabolites were identified (M2 and M3) as its carboxylic acid and hydroxylated ethylester forms, respectively. [18F]flumazenil in vivo nanoPET/CT imag-ing and ex vivo bio-distribution assay also showed significant effects on GABAA receptor availa-bility in the amygdala, prefrontal cortex, cortex, and hippocampus in the rat brain, worriless about the formation of metabolites. We showed completion of the bio-transformed course of flumazenil by the hepatic system; and [18F]flumazenil can be a good ligand and serve as a PET agent for determination of GABAA/BZR complex for multiplex neurological syndromes in the clinical stage.

Background: The official standard for quality control of honey is currently based on physicochemical properties. However, this method is time-consuming, cost intensive, and do not lead to information on the originality of honey. This study aims to classify raw stingless bee honeys by bee species origins as a potential classifier using NMR-LCMS-based metabolomics approach. Methods: Raw stingless bee honeys were analysed and classified by bee species origins using proton nuclear magnetic resonance (1H NMR) spectroscopy and an ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF MS) in combination with chemometrics tools. Results: The honey samples were able to be classified into three different groups based on the bee species origins of Heterotrigona itama, Geniotrigona thoracica, and Tetrigona apicalis. D-Fructofuranose (H. itama honey), β-D-Glucose, D-Xylose, α-D-Glucose (G. thoracica honey), and L-Lactic acid, Acetic acid, L-Alanine (T. apicalis honey) identified via 1H NMR data and the diagnostic ions of UHPLC-QTOF MS were characterized as the discriminant metabolites or putative chemical markers. Conclusion: It could be suggested that the quality of honey in terms of originality and purity can be rapidly determined using classification technique by bee species origins via 1H NMR- and UHPLC-QTOF MS-based metabolomics approach.

Sphingolipids are ubiquitous in eukaryotes and certain prokaryotes, where they serve as vital components of biological membranes and as bioactive molecules. Chloroplasts have complex membrane structures that play crucial roles in photosynthesis, but their specific sphingolipidome remains unreported. In this study, we used high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) to analyze the sphingolipidome of purified Arabidopsis thaliana chloroplasts. We detected 92 chloroplast sphingolipids. The chloroplast sphingolipidome differed from total leaf (TL) samples, having a higher content of free long-chain bases and hydroxyceramides, and a greater proportion of complex sphingolipids with 16C fatty acid (FA) forms. Notably, chloroplast glucosylceramides were predominantly the d18:1 h16:0 and t18:1 h16:0 forms, rather than the 24C FA form found in TL and other cellular structures. Comparing the sphingolipidomes of different cellular structures underscores the inhomogeneity of intracellular distribution of sphingolipids. This provides a robust reference for further elucidating the function of sphingolipids in plant cells.

Plaque psoriasis is a common, long-lasting illness that affects the immune system and causes significant negative impacts on a patient's physical health, well-being, and ability to work effectively. Deucravacitinib (DEU) is the first oral medication used in the treatment of plaque psoriasis, a chronic skin condition that causes red, scaly patches on the skin. DEU is a type of medication called an oral Janus kinase (JAK) inhibitor, which works by blocking specific enzymes that play a role in the inflammation and immune response associated with psoriasis. Therefore, a quick, easy, novel, reliable, sensitive, and straightforward Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) approach was used to analyse DEU in plasma samples. The LC-MS/MS method for the determination of DEU in human plasma was based on using trimethoprim as an internal standard (IS). The separation of DEU and IS was carried out via liquid-liquid extraction (LLE). The isolated substances were then subjected to the chromatographic system using the ACE-C18 column (4.6x100 mm, 5 µm). The mobile phase employed consisted of methanol and a solution of 2 mM ammonium formate (80:20 v/v, respectively). The flow rate used was set at 0.9 mL min-1. The creative strategy was performed by running an ABSCIEX API 4000 mass spectrometer with an electron spray ionization source in Multiple Reaction Monitoring (MRM) modes. The ion transitions m/z 426.3  358.2 was used for DEU quantitation, while the ion transitions m/z 291.1  261.1 was used for trimethoprim quantitation. The accuracy, precision, linearity, recovery, and selectivity of DEU were deemed acceptable when validated for a concentration range between 0.500 to 601.050 ng/mL, utilizing a weighting factor of 1/x2.

Osteoarthritis (OA) is a pathology of great impact worldwide which its physiopathology is not completely known and usually diagnosed by imaging techniques performed at advanced stages of the disease. The aim of this study was to evaluate early serum metabolome changes and identify the main metabolites involved in an inflammatory OA animal model. The study was performed with thirty rats, OA in all animals was induced by intra-articular injection of monoiodoacetate into the knee joint. Blood samples were taken from all animals and analyzed by mass spectrometry before OA induction, and 28, 56 and 84 days following induction. Histological study confirmed OA in all samples. The results of this study allow the identification of several changes in the serum metabolome over time, including organic acids, benzenoids, heterocyclic compounds and lipids at T28, organic acids at T56 and lipid classes at T84. We conclude that OA induces serological changes in the metabolome and, more specifically, 18 metabolites which could serve as potential biomarkers. However, it was not possible to establish a relationship between the identified metabolites and the time at which the samples were taken. Therefore, these findings should be confirmed in future OA studies.

Abstract: (1) Background: No consensus has been reached on indicators and protocols for monitoring drug concentrations in organ transplant recipients for the immunosuppressant mycophenolic acid (MPA) in clinical therapy.In this study, we propose to establish a Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS) for quantitative determination of MPA and its metabolites ( mycophenolic acid carboxybutoxyether, MPAG and mycophenolic acid carboxyglucuronide, AcMPAG) concentrations in peripheral blood mononuclear cells (PBMCs) and to assess the clinical application of PBMCs concentration detection of MPA and its metabolites in infection prediction in Chinese kidney transplant recipients(KTRs); (2) Methods: PBMCs were isolated with Ficoll-Paque solution, and cells were counted. Acetonitrile precipitated proteins to extract the target compounds.Waters ACQUITY UPLC BEH C18 column of 1.7μm (2.1 mm*50 mm) and an ACQUITY-Xevo TQ-S UPLC-MS/MS system were used for the detection with mobile phases: water and methanol (containing 2 mmol/L NH4Ac + 0.1% formic acid); flow rate: 0.3 mL/min. After gradient elution, the samples were positively ionized by an electrospray positive ion source (ESI) and then quantitatively analyzed by a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode. The performance of the established method was evaluated through a series of validation experiments according to the requirements of the relevant guidelines. We enrolled 7 KTRs with stable conditions and 8 KTRs with infection from August to November 2022, who were treated at the West China Hospital of Sichuan University.Their peripheral blood samples were collected and detected MPA, MPAG, and AcMPAG concentrations in PBMCs using the methods mentioned above and evaluated for their clinical application in the prediction and monitoring of infections in KTRs; (3) Results: After validation of the methodology, the UPLC-MS/MS method established in this study for the determination of MPA, MPAG, and AcMPAG concentrations in PBMCs showed good linearity in the quantitative ranges of 0.0625-50 ng/ml, 0.5-50 ng/ml and 0.1-20 ng/ml, and the selectivity, specificity, matrix effect, carry-over contamination, reproducibility, precision and accuracy all satisfied the requirements.AcMPAG in PBMCs was poorly stabilized and showed significant degradation after -80°C storage for 24 h or 3 cycles of freeze-thaw.There was a large variability in MPA and MPAG concentrations in PBMCs of KTRs and it correlated poorly with plasma MPA concentrations (rs=0.206,p=0.117).Subgroup analysis showed that the correlation between MPA in plasma and PBMCs was still poor in the infected group(rs=0.201,p=0.116), but significant correlation in the stable group (rs=0.643,p=0.001). A significantly higher level of MPA in PBMCs was observed in the infected group than those in the stable group at 2h and 4h after drug dosing (p&lt;0.05).AcMPAG concentrations in PBMCs of KTRs were extremely low and virtually undetectable.ROC curve analysis (area under the curve, AUC) was performed for infection after renal transplantation with indicators of significant difference showed that the area AUC for PBMCs-MPA-C4 was 0.928 ,for PBMCs-MPA-C2 was 0.875,and for Plasma-MPA-C4 was 0.803; (4) Conclusions: In this study, a method based on UPLC-MS/MS was developed and validated for the simultaneous determination of MPA, MPAG, and AcMPAG concentrations in PBMCs.With easy operability and good stability, this method provides a reliable analytical method for the clinical implementation of the detection of MPA, MPAG, and AcMPAG concentrations in PBMCs.The concentrations of MPA and MPAG in PBMCs from KTRs were highly variable and were closely associated with the development of infections after renal transplantation.Monitoring MPA and MPAG in PBMCs may be useful indicators for mycophenolic acid therapeutic drug concentration monitoring (TDM) in KTRs.

A convenient and fast multiresidue method for the efficient identification and quantification of 72 pesticides belonging to different chemical classes in red and white grape wines have been developed. The analysis was based on gas chromatography tandem quadrupole mass spectrometric determination (GC–MS/MS). An optimization strategy involved the selection of MWCNTs amount and cleanup procedure cycle times for m-PFC was performed to achieve ideal recoveries and reduce sample matrix compounds in the final extracts. The optimized procedure obtained consistent recoveries between 70.2 and 108.8% (70.2% and 108.8% for white wine, 72.3% and 106.0% for red wine) , with relative standard deviations (RSDs) generally lower than 8.3% at three spiking levels of 0.01, 0.05 and 0.2 mg/kg. Linearity was studied in the range between 0.005 and 0.2 mg/kg using pesticide standards prepared both in pure solvent and in the presence of matrix, showing coefficients of determination (R²) higher than 0.985 for all the pesticides. To improve accuracy, matrix matched calibration curves were used for calculating the quantification results. Finally, the method was used successfully in detecting pesticide residues in commercial gape wines.

Chamazulene (CA) is an intensely blue molecule with a wealth of biological properties. In cosmetics chamazulene is exploited as a natural coloring and soothing agent. CA is unstable and tends to spontaneous degradation accelerated by light. We studied the photodegradation of CA upon controlled exposure to UVB-UVA irradiation by multi-ple techniques, including GC-MS, UHPLC-PDA-ESI-MS/MS and by direct infusion in ESI-MSn, which were matched to in silico mass spectral simulations to identify degra-dation products. Seven byproducts formed upon UVA exposure for 3 h at 70mW/cm2 (blue-to-green color change) were identified, including CA dimers and CA benzenoid which were not found on extended 6 h irradiation (green-to-yellow fading). Photosta-bility tests with reduced irradiance conducted in various solvents in the pres-ence/absence of air indicated highest degradation in acetonitrile in the presence of ox-ygen, suggesting a photo-oxidative mechanism. Testing in the presence of antioxidants (tocopherol, ascorbyl palmitate, hydroxytyrosol, bakuchiol, γ-terpinene, TEMPO and their combinations) indicated highest protection by tocopherol and TEMPO. Sun-screens ethylhexyl methoxycinnamate and particularly Tinosorb® S (but not octo-crylene) showed good CA photoprotection. Thermal stability tests indicated no degra-dation of CA in acetonitrile at 50°C in the dark for 50 days; however, accelerated deg-radation occurred in the presence of ascrorbyl palmitate.

Bat feces were reported in ancient Chinese medicine books to have the effect of reducing heat and improving eyesight, but the mechanism of vision improvement still needs further research. To this end, we used 2,2-diphenyl-1-picrylhydrazyl (DPPH) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) to analyze the antioxidant capacity of and the types of vitamins in bat feces. We hoped to screen the pharmacological components of bat feces, and to explain the role that these components may play in the treatment of visual deterioration. Our results found that bat feces had a good antioxidant capacity and mainly contain vitamins B1 (thiamine), B2 (riboflavin), B3 (nicotinamide), B3 (nicotinic acid), and B5 (pantothenic acid). Although these vitamins may help maintain the health of the optic nerve and cornea, the vitamin content of bat feces is low, but the heavy metal content is high, as shown using inductively coupled plasma mass spectrometry (ICP-MS) analysis. Therefore, we suggest that the use of bat feces as traditional Chinese medicine to improve vision should be strictly restricted.

Reef-dwelling calcifiers face numerous environmental stresses associated with anthropogenic carbon dioxide emissions, including ocean acidification and warming. Photosymbiont-bearing calcifiers, such as large benthic foraminifera, are particularly sensitive. To gain insight into their resistance and adaptive mechanisms to climate change, Amphistegina lobifera from the Gulf of Aqaba were cultured under elevated pCO2 (492, 963, and 3182 ppm) fully-crossed with elevated temperature (28°C and 31°C) for two months. Differential protein abundances in host and photosymbionts amongst treatments were investigated alongside physiological responses and microenvironmental pH variations. Over 1000 proteins were identified, of which one-third varied significantly between treatments. Thermal stress induced protein depletions, along with reduced holobiont growth. Elevated pCO2 caused only minor proteomic alterations and color changes. However, combined stressors reduced pore sizes and increased microenvironmental pH, indicating adaptive modifications to gas exchange. Notably, substantial proteomic variations at moderate-pCO2 and 31°C indicate cellular stress, while stable physiological performance at high-pCO2 and 31°C is scrutinized by putative decreases in test stability. Our experiment shows that the effects of climate change can be missed when stressors are assessed in isolation, and that physiological responses should be assessed across organismal levels to make more realistic predictions for the fate of reef calcifiers.

We have previously shown that 2-thiouridine (S2U), either as a single nucleoside or as an element of RNA chain, is effectively desulfurized under applied in vitro oxidative conditions. Chemically induced desulfuration of S2U resulted in two products: 4-pyrimidinone nucleoside (H2U) and uridine (U). Recently, we investigated whether desulfuration of S2U is a natural process that also occurs in cells exposed to oxidative stress or whether it only occurs in the test tube during chemical reaction with oxidants at high concentrations. Using different types of eukaryotic cells, such as baker's yeast, human cancer cells or modified HEK293 cells with impaired antioxidant system, we confirmed that 5-substituted 2-thiouridines are oxidatively desulfurized in the wobble position of the anticodon of some tRNAs. Quantitative LC-MS/MS-MRMhr analysis of nucleoside mixtures obtained from hydrolyzed tRNA revealed the presence of desulfuration products of mcm5S2U: mcm5H2U and mcm5U modifications. We also observed some amounts of immature cm5S2U, cm5H2U and cm5U products, which may indicate a disruption of an enzymatic modification pathway at the C5 position of 2-thiouridine. The observed process, which is triggered by oxidative stress in living cells, could impair the function of 2-thiouridine-containing tRNAs and alter the translation of genetic information.

In this study, the microbial strain was isolated from the humus soil to ferment Sophora flavescens and Eleutherococcus sessiliflorus extracts. The isolated microbial was identified as Bacillus genus by 16S rRNA sequence analysis. The fermented plant extracts exhibited the antifungal effects against four types of the plant pathogens such as P. carotorum, B. cinerea, C. fructicola Sau-3, and C. gloeosporioides according to incubation time. Especially, the fermented plant extracts showed the most active activities for Colletotrichum genus to inhibit mycelium growth. Metabolite changes in fermented S. flavescens and E. sessiliflorus extracts were confirmed through LC-Q-TOF/MS. Flavonoid and peptide derivatives were improved in fermented S. flavescens and E. sessiliflorus extracts compared to their unfermented one, respectively. This study suggested that the isolated Bacillus microbial fermentation could be a valuable tool in improving the bioactivity of S. flavescens and E. sessiliflorus extracts to have potential with more environmentally-friendly antifungal agents.

Alterations in the carnitine shuttle system may be an indication of the presence of cancer. As such, in-depth analyses of this pathway in different malignant tumors could be important for the detec-tion and treatment of this disease. The current study aims to assess the profiles of carnitine and acylcarnitines in gliomas with respect to their grade, the presence of isocitrate dehydrogenase (IDH) mutations, and 1p/19q co-deletion. Brain tumors obtained from 19 patients were sampled on-site using solid-phase microextraction (SPME) immediately following excision. Analytes were desorbed and then analyzed via liquid chromatography-high resolution mass spectrometry. The results showed that SPME enabled the extraction of carnitine and 22 acylcarnitines. An analysis of the correlation factor revealed presence of two separate clusters: short-chain and long chain car-nitine esters. Slightly higher carnitine and acylcarnitine concentrations were observed in the higher malignancy tumor samples (high vs low grade), and in those samples with worse projected clinical outcomes (without vs with IDH mutation; without vs with 1p/19q co-deletion). Thus, the proposed chemical biopsy approach offers a simple solution for on-site sampling that enables sample preservation, thus supporting comprehensive multi-method analyses.

A novel determination method using flow-through cartridge purification and ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was developed for the identification and quantification of 30 mycotoxins (aflatoxin B1, B2, G1, G2, sterigmatocysin, T-2 toxin, diacetoxyscirpenol, roquefortine C, methylergonovine, ergocornine, lysergol, enniatin A, enniatin A1, enniatin B, enniatin B1, beauvericin, deoxynivalenol, 3-acetylDeoxynivalenol, 15-acetylDeoxynivalenol, patulin, verruculogen, neosolaniol, gliotoxin, HT-2 toxin, wortmannin, zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol) in feedstuffs. Mycotoxins were extracted from sample by 0.1% formic acid aqueous solution/acetonitrile (16/84 v/v), then purified by a Cleanert® MC clean-up column. As a result, the pigments and other matrices were efficient removed from the sample extract solution. The purified eluate was collected, then evaporated and redissolved by 0.1% formic acid aqueous solution/acetonitrile (70:30 v/v) for UPLC-MS/MS detection. A BEH Shield RP18 chromatographic column was employed for separation of 30 mycotoxins in a total of 14 min. Multiple-reaction monitoring (MRM) mode was applied for qualitative and quantitative analysis, and matrix calibration curves obtained with the external-standard method was used for quantitation of target analytes. Under optimized conditions, the linearity range was from 2 to 1000 ng/g, and the limit of quantification of the developed method was from 2 to 50 ng/g. The recoveries of 30 mycotoxins spiked in urine samples were from 72.0% to 118.5%, and the relative standard deviation was below 20%. The method was also well approved by certified reference sample, and applied on the real feedstuff samples testing successfully.

This study developed and validated a robust UPLC-MS/MS method for quantifying cyclic glycine-proline (cGP) in mangrove -derived Penicillium and Aspergillus strains. The method demonstrated excellent linearity, precision, and recovery, with detection limits as low as 4.8 ng/mL. When applied to fungal extracts, Penicillium pedernalense achieved 67.45 1.11 ng/mL (yielding 29.31 0.61 mg/L), surpassing Penicillium steckii at 31.71 0.31 ng/mL (yielding 8.51 0.15 mg/L). This quantitative approach for metabolite analysis provides a viable method for screening these fungal strains, highlighting their potential for sustainable production of cyclic glycine-proline (cGP).

Plants, among which pumpkins (Cucurbita spp.), represent a good source of nutrients and bioactives with interesting health-promoting properties. In this research, carotenoid extracts obtained from the pulp of eight pumpkin varieties, belonging to the C. moschata and C. maxima species, were tested for cytotoxicity on SH-SY5Y neuroblastoma cells. The results showed that pumpkin bioactives exert a cytotoxic action against the tested cells, in particular Butternut extract at 100 μmol/L concentration after 24h of treatment and Mantovana extract at 50 μmol/L after 48 h. Moreover, the carotenoid extracts also showed interesting in vitro antioxidant activity. To fully characterize the qualitative and quantitative profile of carotenoids in the tested extracts, a high-performance chromatographic technique was performed, revealing that pumpkin pulp carotenoids were mainly represented by β-carotene, mono- and di- esterified hydroxy- and epoxy- carotenoids. Moreover, the carotenoid dataset was also useful for discriminating samples from two different species. In conclusion, the results of the present study highlight the intriguing potential of pumpkin carotenoids in the development of related anticancer drugs and chemotherapeutic adjuvants.

Turmeric, Curcuma longa L., is a type of medicinal plant characterized by its perennial nature and rhizomatous growth. It is a member of the Zingiberaceae family and is distributed across the world’s tropical and subtropical climates, especially in South Asia. Its rhizomes are highly valued for food supplements, spices, flavoring agents, and yellow dye in South Asia since ancient times. It exhibits a diverse array of therapeutic qualities that encompass its ability to combat diabetes, reduce inflammation, act as an antioxidant, exhibit anticancer properties, and promote anti-aging effects. In this study, organic extracts of C. longa rhizomes were subjected to HPLC separation followed by mass spectrometry analysis. The Global Natural Product Social Molecular Networking (GNPS) approach was utilized for the first time in this ethnobotanically important species to conduct an in-depth analysis of its metabolomes based on their fragments. A total of 30 metabolites including 16 diarylheptanoids, 1 diarylpentanoid, 3 bisabolocurcumin ethers, 4 sesquiterpenoids, 4 cinnamic acid derivatives, and 2 fatty acid derivatives were identified. Among 16 diarylheptanoids identified in this study, five of them are reported for the first time in this species.

Soybean, maize and bean are crops of great economic importance, but in the last years suffered with infestations of the caterpillar Helicoverpa armigera, being the main problem the resistance of this pest to most pesticides. Avermectin emamectin benzoate was recently released to control this pest. Other avermectins, like abamectin, doramectin, eprinomectin and ivermectin are used in large scale because they potent acaricidal, anthelmintic, and insecticidal activities. Thus, a simple and fast method for the determination of avermectins in these crops based on a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure and ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) analysis was developed and validated. For extraction, water followed by acetonitrile:isopropanol and a partition step with salts was stablished. With the clean-up step using activated EMR-Lipid, limits of detection of 1.2 μg kg-1 for abamectin, doramectin, emamectin benzoate and ivermectin, and of 2.4 μg kg-1 for eprinomectin were achieved. Accuracy and precision evaluated at low levels presented satisfactory results. The method was successfully applied in commercial samples and is a good alternative for routine analysis.

Gold clusters protected by 3-MBA ligands (MBA = mercaptobenzoic acid, -SPhCO2H) have attracted recent interest for their unusual structures and advantageous ligand-exchange and bioconjugation properties. Azubel et al. first determined the core structure of an Au68-complex, which was estimated to have 32 ligands (3-MBA groups). To explain the exceptional structure-composition and reaction properties of this complex, and its larger homologs, Tero et al. proposed a “dynamic stabilization” via carboxyl O-H--Au interactions. Herein, we report the first results of an integrated LC/MS analysis of unfractionated samples of gold / 3-MBA clusters, spanning the narrow size range 13.4 to 18.1 kDa. Using high-throughput procedures adapted from bio-macromolecule analyses, we show that integrated capillary HPLC-ESI-MS, based upon aqueous-methanol mobile phases and ion-pairing reverse-phase chromatography, can separate several major components from the nanoclusters mixture that may be difficult to resolve by standard native gel electrophoresis due to their similar size and charge. For each component, one obtains a well-resolved mass spectrum, nearly free of adducts or signs of fragmentation. A consistent set of molecular mass determinations is calculated from detected charge-states tunable from 3- (or lower), to 2+ (or higher). One thus arrives at a series of new compositions (n, p) specific to the Au/3-MBA system. The smallest major component is assigned to the previously unknown (48, 26); the largest one is evidently (67, 30), vs. the anticipated (68, 32). Various explanations for this discrepancy are considered. A prospective is given for the several members of this novel series, along with a summary of the advantages and present limitations of the micro-scale integrated LC/MS approach to characterize such metallic-core macro-molecules, and their derivatives.

Tamarix nilotica (Ehrenb.) Bunge (Tamaricaceae), an indigenous plant to the Middle East region, is well-known as a medicinal plant for treating many human ailments. The current study aimed at exploring the polyphenols profile of T.nilotica alcohol soluble fraction of aqueous extract, assessing its in-vivo antifibrotic activity and the possible underlying mechanism, to unravel the impact of quantitative difference of sulphated polyphenols content on the antifibrotic activity of T.nilotca grown in two different habitats. Polyphenols profiling of T. nilotica extracts was performed using HPLC-HRESI-QTOF-MS-MS. The major polyphenol components included sulphated flavonoids, phenolic acids and free aglycones. The antifibrotic activity was evaluated through carbon tetrachloride-induced liver fibrosis in rats. Biochemical evaluations revealed that both fractions ameliorated the increased levels of hepatic aminotransferases, lipid peroxidation, hydroxyproline, α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB). Moreover, both fractions reduced catalase activity (CAT) and enhanced hepatic glutathione (GSH) content. Histopathological imaging undoubtedly confirmed such results. In conclusion, T. nilotica polyphenols rich fraction exhibited potential antifibrotic activity in rats. Significant alterations in GSH levels were recorded based on sulphated polyphenol metabolites content.

Re-investigation of the chemical composition of the annual plant Mitracarpus scaber Zucc. led to the identification of clarinoside, a new pentalogin derivative containing a rare quinovose moiety, and the known compound harounoside. While the planar structure was fully determined using tandem MS and quantum mechanics calculations (QM), the tridimensional structure was unravelled after isolation and NMR analysis. The absolute configuration was assigned by comparison of experimental and theoretical SRCD spectra. Both compounds were tested for anti-inflammatory activity and compound 1 showed the ability to inhibit the production of interleukin-8 (Il-8) with an IC₅₀ of 9.17 µM.

Solanum nigrum fruits have been conventionally available as a material of beverage due to its nutritional substances such as minerals, vitamins, amino acids, proteins, sugars, polyphenols and anthocyanins. Here has rarely reported on the characterizaton of the components and the regulatory mechanism of Anthocyanins in S. nigrum. In this study, we determined that the peel and flesh of S. nigrum fruits shared the similar HPLC profiles, but the different contents and total antioxidant activities for Anthocyanins. After an efficient purification method mainly including extraction with pH 1.0 distilled water and then desorption with pH 1.0 95% ethanol after a DM-130 resin adsorption step to obtain more pure anthocyanins extracts, the purity of anthocyanins extract from S. nigrum fruits reached to 56.1%. Moreover, eight anthocyanins from S. nigrum fruit were identified with HPLC-MS/MS for the first time. A typical R2R3-MYB transcription factor gene, SnMYB, was also cloned for the first time by RACE-PCR from S. nigrum. Moreover, the contents of anthocyanins was shown a good correlation (r = 0.93) with the expression levels of SnMYB during the fruits developmental stages. Most significantly, SnMYB successfully produced high anthocyanins contents (1.03 mg/g) when SnMYB was transiently expressed in tobacco leaves. Taken together, S. nigrum fruits are a promising resource for anthocyanins extraction and SnMYB is an activator that positively regulates anthocyanins biosynthesis in S. nigrum.

Metabolomics analysis generates vast arrays of data, necessitating comprehensive workflows involving expertise in analytics, biochemistry and bioinformatics, in order to provide coherent and high-quality data that enables discovery of robust and biologically significant metabolic findings. In this protocol article, we introduce NoTaMe, an analytical workflow for non-targeted metabolic profiling approaches utilizing liquid chromatography–mass spectrometry analysis. We provide an overview of lab protocols and statistical methods that we commonly practice for the analysis of nutritional metabolomics data. The paper is divided into three main sections: the first and second sections introducing the background and the study designs available for metabolomics research, and the third section describing in detail the steps of the main methods and protocols used to produce, preprocess and statistically analyze metabolomics data, and finally to identify and interpret the compounds that have emerged as interesting.

Vitamin D is crucial for athletes' health and performance. Although the exact vitamin D requirements for athletes have not been established, maintaining 25(OH)D levels of at least 40 ng/ml is considered beneficial. This study aimed to determine whether an individual loading dose formula for vitamin D supplementation is suitable to meet a target value of 40 ng/ml and is more effective than standardized supplementation. In a 10-week supplementation study during the winter months in Germany, 90 athletes with insufficient vitamin D levels (25(OH)D<30ng/ml) were randomly assigned to receive either a universal dose of 2000 IU/day of vitamin D or a loading dose of 4000 IU/day, followed by a maintenance dose of 1000 IU/day. Total 25(OH)D concentration was measured from dried blood spots at three time points: at baseline, at the computed date of 40ng/ml, and after the 10-week period. Additionally, a vitamin D-specific questionnaire was issued. On the day when 25(OH)D blood concentrations of 40 ng/ml were calculated to prevail, the individualized group had a significantly higher 25(OH)D level than the standardized group (41.1+-10.9 ng/ml vs. 32.5+-6.4 ng/ml, p<0.001). This study demonstrated that the examined formula is suitable for athletes to achieve a 25(OH)D concentration of 40 ng/ml. This indicates that a personalized approach is more effective than a one-size-fits-all approach in restoring adequate vitamin D levels in athletes.

In this study, a new approach to pesticide permeation through the apple peel into the pulp is discussed. The tested compounds can be classified, based on mode of action, as systemic (boscalid, cyprodinil, pirimicarb, propiconazole and tebuconazole) or contact (captan, cypermethrin and fludioxonil) pesticides. The barrier effect was assessed using a Franz flow-type vertical diffusion cell system. Residue analysis was performed using a modified quick, easy, cheap, efficient, rugged and safe (QuEChERS) extraction method coupled to gas chromatography with tandem mass spectrometry (GC-MS/MS). The limits of detection (LODs) ranged between 2.6 µg kg-1 (pirimicarb) and 17 µg kg-1 (captan), with the coefficient of variability (CV) lower than 6%, while recoveries ranged from 85% (boscalid) to 112% (captan) at 0.1 and 1 mg kg-1 spiked levels. The highest peel penetration was observed for pirimicarb, captan and cyprodinil, with cumulative permeation of 90, 19 and 17 µg cm-2, respectively. The total absorption was in the range from 0.32% (tebuconazole) to 32% (pirimicarb). Only cypermethrin was not quantitatively detected in the pulp and its use can be recommended in crop protection techniques. The obtained results indicate that molecular weight, octanol-water partition coefficient and water solubility are important parameters determining the process of pesticide absorption.

Wheat is one of the global strategic crops and ranks third in terms of cereals production. Wheat crop is exposed to many fungal infections during the cultivation stages, some of which have the ability to secrete numbers of toxic secondary metabolites that threaten the quality of grains, consumer health, producer economics and global trade exchange. Fifty-four random samples were collected from wheat which originated from different countries. The samples included 14 of soft wheat to study the extent of their contamination with Deoxynivalenol (DON) and T-2 toxin by auto-ELISA technology and r-biopharm Microtiter plate. All samples were contaminated with DON toxin except one sample, and the contamination average ranged between 40.7 and 1018.8 µg kg-1. The highest contamination rates were in Lithuanian and the lowest in Indian wheat. Meanwhile the highest average level of T-2 toxin contamination was in Lithuanian wheat grains with a rate of 377.4µg kg-1 and the lowest in Polish wheat with an average of 115.3µg kg-1. GC-MS/MS and multiple reaction monitoring mode (MRM), was used to detect 15 triazole derivatives in collected samples, which may be used to combat fungal diseases on wheat during the growing season. Only 9 derivatives were found; Simeconazole, Penconazole, Hexaconazole, Cyproconazole, Diniconazole, Tebuconazole, Metconazole, Fenbuconazole and Difenoconazole. These derivatives varied according to the origin of wheat samples as well as their its concentration whereas, another 6 derivatives were not detected in all samples. A direct inverse relationship was found between the DON concentration in the samples and the residues of Simeconazole, Penconazole, Diniconazole, Tebuconazole, Metconazole, Fenbuconazole and Difenoconazole and the T-2 toxin showed the same relationship except for Tebuconazole. The safe and rational use of some triazole derivatives may be a new approach and a promising strategy not only to reduce plant diseases and their problems, but also to get rid of some mycotoxins as contaminates.

Andean blueberries are wild berries grown and consumed in Ecuador which contain high values of bioactive compounds, mainly anthocyanins, with powerful antioxidant activity. The aim of this study was to evaluate the profile and contents of (poly)phenols and carotenoids in Andean blueberry by HPLC-DAD-MSn and determine a wide range of its biological activities. The antioxidant capacity of this fruit was evaluated in vitro by three different methods and in vivo using the zebrafish animal model, also the toxicity effect was determined by the zebrafish embryogenesis test. Besides, the antimicrobial activity and the capacity of Andean blueberry to produce hemagglutination in blood cells were evaluated. Finally, the bioaccessibility of (poly)phenols and related antioxidant capacity were determined in the different phases of an in vitro digestion. The global results indicated no toxicity of Andean blueberry, weakly bacteriostatic activity, and high contents of anthocyanins and antioxidant capacity, which were partially bioaccesible in vitro (~ 50 % at the final intestinal step), contributing to the knowledge of its health benefits for consumers and its potential use in the food and pharmaceutical industry as functional ingredient.

The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiate them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled comprehensive analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids and proteins in cell culture is challenging due to the compound’s chemical difference so that most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two–phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C-isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS) and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over 3-4 orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0) and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications.

Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major economically important legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Obviously, changes in seed protein patterns might directly affect both of these aspects. Thus, here we address the pea seed proteome in more detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. Accordingly, 1938 and 1989 non-redundant proteins were identified in yellow and green pea seeds, in total. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP) potentially indicating the high efficiency of our experimental workflow. In total 981 protein groups could be assigned to 34 functional classes, which were to a large extent differentially represented in yellow and green seeds. Closer analysis of these differences by processing of the data in KEGG and String databases revealed their possible relation to a higher metabolic status and reduced longevity of green seeds.

Considering the prevalence of cropping pattern in many Saffron cultivation regions, the continuous cropping obstacle caused the decline in growth and development. These effects justify the necessity of studying agronomic strategies to promote plant quality and production. Herein, the mono cropping, intercrop of Paulownia and Saffron, and intercrop of Soybean and Saffron patterns have been performed. The metabolites of Saffron under three cropping patterns were determined and identified using by an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Uni and multivariate statistical analysis were applied to examine the difference between groups and select the significant differential accumulated metabolites. The results showed that a total of 315 compounds were significantly changed in three comparison groups. Phenylalanine metabolism, linoleic acid metabolism, and flavonoid biosynthesis pathway has been greatly enriched by the changed metabolites. The phenyl lactate, trans-2-Hydroxy-cinnamate, γ-Linolenate, 9-oxo-octadecadienoic acid, 13-0xo-0ctadecadienoic acid, 9,12,13-TriHOME of those metabolites showed significant differential accumulation in Saffron under three cropping patterns. And the accumulation of safranal and picrocrocin related compounds, such as Crocetin, Crocin showed a similar variation regularity. These indicate that there may be a close internal relationship between the cropping pattern and the biosynthesis of metabolites related to endogenous substances and Saffron quality formation.

Cathinone is a natural stimulant found in Catha edulis and commonly used in new psychoactive substances. The objective of this study was to examine the disposition kinetics and metabolic profile of cathinone and its metabolite cathine through a single oral dose of cathinone administration in rats. Cathine and cathinone concentrations were identified and quantified using ion trap liquid chromatography (LC-IT/MS), while the metabolic profile was determined using the quadrupole time of flight UPLC-QTOF/MS method in the serum, brain, lung, liver, kidney, and heart over a period of 0, 0.5, 2.5, 6, 12, 24, 48, and 72 hours. The findings revealed a dynamic interplay between the pharmacokinetics of cathinone and its impact on organ-specific biomarkers. The distribution of cathinone and cathine was determined in serum, brain, heart, lung, kidney, and liver. The highest concentration of cathinone was found in the kidney at 1438.6 ug/L. It gradually decreased to 1.97 within 48 hours and disappeared after 72 hours. Within half an hour, cathinone levels in the lungs, liver, and heart were found to be 859, 798.9, and 385.8 ug/L, respectively. However, within 2.5 hours, these levels decreased to 608.1, 429.3, and 309.1 ug/L and kept decreasing until they became undetectable after 24 hours. The concentration of cathinone in the rat brain reduces quickly, dropping to undetectable levels within six hours, with levels decreasing from 712.7 ug/L after just 30 minutes. In the brain and serum, cathine reached its highest levels at 2.5 hours, while in other organs, it peaked at 0.5 hours. This indicates that the conversion of cathinone to cathine is slower in the brain and serum. The study found that cathinone's pharmacokinetics have a significant impact on organ-specific biomarkers. After administering the cathinone, there was a sharp change in biomarker levels, particularly in the liver and brain, during the initial hours. It is essential to investigate the correlation between the changes in biomarkers found in the brain and the levels of cathinone and cathine. The results have significant implications for drug development, pharmacovigilance, and clinical practices involving cathinone. In conclusion, this comprehensive analysis of cathinone's impact on organ-specific biomarkers provides a basis for informed decision-making in medical practices and further research into the cathinone's pharmacological properties.

The aim of this study was to investigate the parameters affecting the extraction of positive molecules such as chlorogenic acids and antioxidants, as well as potentially carcinogenic substances such as acrylamide, in different coffee brewing methods. Three coffee varieties, each assigned to different roasting degree, were used to prepare coffee brews following eight diverse preparation methods. Acrylamide was quantified by HPLC-MS/MS instrument while chlorogenic acids and caffeine by HPLC-DAD system. Three spectrophotometric analyses were also performed (DPPH, TFC and TPC) to evaluate antioxidant activity as well. The results showed that the main brewing parameters, that have the greatest influence on the final content of these compounds were the volume of water used, more specifically the brewing ratio (coffee to water ratio), the extraction time and the particle size of the coffee powder.

Propolis was widely used in health preservation and disease healing, it contains many ingredients. The previous study had been revealed that the propolis has a wide range of efficacy, such as antiviral, immune enhancement, anti-inflammatory and so on, but its antiviral components and underlying mechanism of action remain unknown. In this study, we investigated the chemical composition, and anti-PPV and immunological enhancement of Propolis Flavonoid(PF). Chemical composition of PF was distinguished by UPLC-Q/TOF-MS/MS analysis.The presence and characterized of 26 major components was distinguished in negative ionization modes.To evaluate the effects of PF used as adjuvant on the immune response porcine parvovirus (PPV). Thirty Landrace-Yorkshire hybrid sows were randomly assigned to 3 groups, and the sows in adjuvant groups were intramuscular injected PPV vaccine with 2.0 mL PF adjuvant (PA), oilemulsion adjuvant (OA), respectively. After that, serum hemagglutination inhibition antibody titers, IgM and IgG subclasses, eripheral lymphocyte proliferation activity, and concentrations of cytokines were measured. Results indicated an enhancing effect of PA on IgM, IL-2, IL-4, IFN-γ and the IgG subclass responses. These findings suggested that PA could significantly enhance the immune responses. Furthermore, we screened the chemical components the effective of anti-PPV, Ferulic acid have an excellently anti-PPV effective.

Microsamples (collections usually less than 50 µL) have been introduced in pre-clinical, clinical, and research settings to overcome obstacles in sampling via traditional venipuncture. However, venipuncture remains the sampling gold standard for metabolic phenotyping of blood. This pre-sents several challenges in metabolic phenotyping workflows: accessibility for individuals in ru-ral and remote underserved areas (due to the need for trained personnel), the unamenable nature to frequent sampling protocols in longitudinal research (for its invasive nature), and sample col-lection difficulty in the young and elderly. Furthermore, venous sample stability may be compro-mised when temperate conditions necessary for cold-chain transport are beyond control. Alter-natively, research utilising microsamples extends phenotyping possibilities to inborn errors of metabolism, therapeutic drug monitoring, nutrition, as well as sport and anti-doping. Although the application of microsamples in metabolic phenotyping exists, it is still in its infancy, with whole blood being overwhelmingly the primary biofluid collected through the collection method of dried blood spots. Research into metabolic phenotyping of microsamples is limited; however, with advances in commercially available microsampling devices, common barriers such as volumetric inaccuracies and the ‘haematocrit effect’ in dried blood spot microsampling can be overcome. In this review, we provide an overview of the common uses and workflows for mi-crosampling in metabolic phenotyping research. We discuss the advancements in technologies, highlighting key considerations and remaining knowledge gaps for employment of microsamples in metabolic phenotyping research. Supporting the translation of research from the ‘bench to the community’.

No study has focused on Uvaria macrophylla leaves with various traditional efficiencies. This paper therefore applied a database-affinity ultra-high-performance liquid chromatography with quadrupole orbitrap tandem mass spectrometry (UHPLC‑Q‑Orbitrap‑MS/MS) strategy, to analyze the lyophilized aqueous extract of U. macrophylla leaves. Through database comparison and MS fragment elucidation, the study has putatively identified 41 constituents belonging to flavonoid, phenolic acid, steroid, saccharide natural product classifications. Significantly, 4 groups of isomers (liquiritigenin vs isoliquiritigenin vs pinocembrin; oroxylin A vs wogonin vs galangin 3-methyl ether; isoquercitrin vs hyperoside; protocatechuic acid vs 2,5-dihydroxybenzoic acid) have been successfully distinguished from each other. All of 41 constituents were then subjected to a quantitative analysis based on linear regression equation established by the above UHPLC‑Q‑Orbitrap‑MS/MS strategy, and an ABTS+•-scavenging antioxidant assay. Finally, the chemical content was multiplied by the corresponding ABTS+•-scavenging percentage to calculate the antioxidant contribution. It was shown that the chemical contents of 41 constituents varied from 0.003 ± 0.000 to 14.418 ± 1.041 mg/g, and gallic acid showed the highest antioxidant contribution. Gallic acid is considered as a suitable antioxidant quality-marker (Q-marker) of U. macrophylla leaves. These findings have scientific implications for resource development and quality-control of U. macrophylla leaves.

The natural compounds of boron have many applications, including as dietary supplement. Communication is based on the discovery that diester chlorogenoborate complex can be detected and quantified from green coffee bean. The study reports that such diester molecule can also be synthesized in stable form by direct reaction of boric acid and chlorogenic acid, in a mixture of acetonitrile–water (1:1, v/v), left to evaporate over a period of 48 hours, at room temperature, resulting a spirocyclic form (diester complex). The diester complex, with its molecular structure and digestibility attributes has potential application as prebiotic in gut health, oral health and as micronutrient essential for microbiota to human and animals.

The “circular bioeconomy” offers numerous opportunities for the health, cosmetic, and nutrition sectors through the potential reuse of Castanea sativa by-products. This abundant and sustainable resource provides valuable bioactive secondary metabolites with antioxidants and anti-inflammatory properties. The transformation of these by-products into high-value products for human health can contribute to sustainable economic growth and reduce the environmental impact of traditional waste disposal practices, thus creating added value from previously underutilized resources. In the present study, we investigated the antioxidant capacity, phytochemical composition and in vitro antioxidant and anti-inflammatory activity of C. sativa burrs (CSB) aqueous extract. The spectrophotometric study yielded optimal total phenolic content (TPC) values, along with significant antioxidant and anti-radical properties. The utilization of UPLC-MS-MS techniques for phytochemical investigation resulted in the identification of a total of 56 metabolites, providing confirmation of the presence of phenolic compounds in CSB. In addition, CSB significantly downregulated pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophage cells without significant cell toxicity.

Traditional Filuferru is an ancient spirit from Sardinia, Italy, usually obtained from the distillation of wine or grape marc. In this contribution, the results of the first chemical characterization of a wide number of craft Filuferru samples has been accomplished in terms of evaluation of the alcoholic strength, qualitative and quantitative GC-MS analysis of the volatile composition of the distillate, and its trace element composition by means a ICP-MS method. Both instrumental methods have been validated and applied on 21 craft samples of Filuferru, whereas one sample of commercial distillate has been analyzed for comparison purposes. Alcoholic strength ranged between 41.0 and 62.4% (v/v). Sixty volatile compounds were identified and ten of them have been quantified. Analogies and differences with Grappa (i.e. the Italian distilled spirit most close to Filuferru) have been highlighted in the qualitative and quantitative profile of this matrix. Often meaningful amounts of acetaldehyde, ethyl acetate, dietyl acetal and acetic acid were measured. Elemental analysis, performed on toxic, non-toxic elements and oligoelements, 18 in total, revealed a wide variability of concentrations in both analytes and samples. High concentrations of Cu are sometimes evidenced, likely caused by losses from the distillation apparatus. The principal components analysis (PCA) allowed the differentiation of the ten volatile compounds quantified in two groups: the former, described mainly by PC1, constituted by acetic acid, ethyl acetate, dietyl acetal and acetaldehyde, and the second, described by PC2, constituted by 1-propanol, 2-methyl-1-propanol, the two coeluiting isomers 2-methyl-1-butanol and 3-methyl-1-butanol,1-hexanol, 2-phenylethanol and 2,3-butanediol. Data obtained may be useful in order to establish a regulation for the production of high-quality traditional Filuferru from Sardinia.

Brain tumors are neoplasms with one of the highest mortality rates. Therefore, the availability of methods that allow the quick and effective diagnosis of brain tumors and selection of appropriate treatments is of critical importance for patient outcomes. In this study, coated blade spray-mass spectrometry (CBS-MS), which combines the features of microextraction and fast ionization methods, is applied for the analysis of brain tumors. In this approach, a sword-shaped probe is coated with a sorptive material to enable the extraction of analytes from biological samples. The analytes are then desorbed using only a few microliters of solvent, followed by the insertion of the CBS device into the interface on the mass spectrometer source. The results of this proof-of-concept experiment confirmed that CBS coupled to high-resolution mass spectrometry (HRMS) enables the rapid differentiation of two histologically different lesions: meningiomas and gliomas. Moreover, quantitative CBS-HRMS/MS analysis of carnitine, the endogenous compound, previously identified as discriminating metabolite showed good reproducibility with the variation below 10% when using a standard addition calibration strategy and deuterated internal standards for correction. The resultant data show that the proposed CBS-MS technique can be useful for on-site qualitative and quantitative assessments of brain tumor metabolite profiles.

Multiple Sclerosis (MS) is a progressive neurodegenerative disease that affects more than 2 million people worldwide. Increasing knowledge about MS in different populations has advanced our understanding of disease epidemiology and variation in the natural history of MS among White and minority populations. In addition to differences in incidence, African American (AA) and Hispanic patients have greater disease burden and disability in earlier stages of disease compared to White patients. To further characterize MS in AA and Hispanic populations, we conducted a retrospective chart analysis of 112 patients treated at an MS center in Houston, Texas. Here, we describe differences in clinical presentation, MRI findings, treatment regimens, disability progression, and relapse rate. We found that patients who were evaluated by a neurologist at symptom onset had significantly decreased odds of greater disability [defined as Expanded Disability Status Scale (EDSS) &gt; 4.5] at last presentation compared to patients who were not evaluated by a neurologist (OR: 0.04, 95% CI: 0.16-0.9). We also found that active smokers had significantly increased odds of greater disability both at diagnosis and at last clinical encounter compared to nonsmokers (OR: 2.44, 95% CI: 1.10-7.10, OR= 2.44, 95% CI: 1.35-6.12, p= 0.01, respectively). Assessment of the degree of brain atrophy and progression over time along with enumeration of T1, T2, and gadolinium-enhancing brain lesions did not reveal differences across groups.

This study assessed the impact of inoculation with plant growth-promoting bacteria (PGPB) on stress-resistance and growth-promoting metabolites in Salicornia europaea L. across controlled and field conditions. Salicornia europaea seeds, inoculated with Brevibacterium casei EB3 and Pseudomonas oryzihabitans RL18, were grown in controlled laboratory experiments and in a nat-ural field setting. Metabolite composition was analyzed using GC-MS and UHPLC-MS. Under controlled conditions, PGPB inoculation significantly increased levels of unsaturated fatty acids, sugars, citric acid, acetic acid, chlorogenic acids, and quercetin. Inoculation under field condi-tions increased glucose, fructose, quercetin, and apigenin, alongside decreased unsaturated fatty acids and sterols, indicating a stress response shift in plant metabolism. Under controlled condi-tions, enhanced metabolite profiles induced by inoculation likely contributed to augmented bio-mass production and environmental stress resistance. Field plants revealed distinct phytochemi-cal profiles, suggesting higher stress levels. However, the accumulated metabolites from inocula-tion could enhance the plant's ability to cope with adverse conditions and improving stress tol-erance. This study unveils the intricate metabolic adaptations of Salicornia europaea under con-trolled and field stress conditions, highlighting PGPB's potential to enhance plant stress tolerance. Elevated stress-related metabolites may fortify plant defense mechanisms, laying the ground-work for stress-resistant crop development through PGPB-based inoculants, especially in saline agriculture.

Barley is rich in phenolic compounds, providing health benefits and making it a valuable addition to a balanced diet. However, most studies focus on these compounds at barley's final maturity, neglecting their synthesis during grain development and its impact on barley quality for food applications. This study investigates phenolic profiles during grain development in four hull-less barley genotypes with different grain colors, specifically bred for food applications. The objectives were to determine the phenolic profile and identify the optimal maturity stage for maximum phenolic content and antioxidant capacity. Using UPLC-MS/MS and in-vitro antioxidant capacity assays, results show that total phenolic compounds decrease as grain matures due to increased synthesis of reserve components. Flavan-3-ols, phenolic acids, and flavone glycosides peaked at immature stages, while anthocyanins peaked at physiological maturity. The harvest stage had the lowest phenolic content, with a gradient from black to yellow, purple, and blue genotypes. Antioxidant capacity fluctuated during maturation, correlating positively with phenolic compounds, especially bound phenolic acids and anthocyanins. These findings suggest that early harvesting of immature grain can help retain bioactive compounds, promoting the use of immature barley grains in foods. To support this market, incentives should offset costs associated with decreased grain weight.

Tumor necrosis factor (TNF) is a pleiotropic cytokine regulating many physiological and pathological immune-mediated processes. Although initially described as a tumor-cytotoxic agent, over time, it has also been recognized as an essential pro-inflammatory cytokine implicated in the pathogenesis of different autoimmune diseases, such as multiple sclerosis (MS). Several studies have shown an increase in TNF expression both in acute and chronic active MS brain lesions, as well as its involvement in maintaining a chronic inflammatory intrathecal process known as “compartmentalized inflammation”, which contributes to disease progression and disability accumulation in MS, even more than acute inflammatory activity. Indeed, high TNF levels were observed in the serum and cerebrospinal fluid of subjects with MS and correlated to disease severity, promoting axonal damage and, consequently, neurodegeneration. In this review we discuss the current knowledge of TNF and its receptors involvement in MS progression, focusing on disability progression independent of relapse activity (PIRA), meant as the disability accumulation evident in relapsing MS patients not related to acute inflammatory events.

Cardiovascular diseases (CVD) are the leading cause of premature death and disability in humans. Increasing data suggest that CVD is closely related to lipid metabolism and signaling. This study aimed to assess whether circulating lysophospholipids (LPL), lysophosphatidic acids (LPA) and monoacylglycerols (MAG) may be considered as biomarkers of CVD. For this objective, the evolution of the plasma levels of 22 compounds (13 LPL, 6 LPA and 3 MAG) was monitored by liquid chromatography coupled with tandem mass spectrometry (HPLC/MS²) in different rat models of CVD, i.e. angiotensin-II-induced hypertension (HTN), ischemic chronic heart failure (CHF) and sugen/hypoxia(SuHx)-induced pulmonary hypertension (PH). On one hand, there was modest changes on the monitored compounds in HTN (LPA 16:0, 18:1 and 20:4), LPC 16:1) and CHF (LPA 16:0, LPC 18:1 and LPE 16:0 and 18:0) models compared to control rats but these changes were no longer significant after correction for multiple testing. On the other hand, PH was associated with important changes in plasma LPA with a significant increase in the 16:0, 18:1, 18:2, 20:4 and 22:6 species. A deleterious impact of LPA was confirmed on isolated human pulmonary smooth muscle cells with an increase in their proliferation. This study demonstrates that circulating LPA species are increased in rats with PH and may contribute to the pathophysiology of this disease. Additional experiments are needed to assess whether the modulation of LPA signaling in PH may be of interest.