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Τhyreostats (TSs) are veterinary drugs used in livestock farming for fattening. Their administration is banned in the European Union since 1981, and their monitoring for food quality and safety control requires sensitive and confirmatory methods. The present study describes the development and validation of a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6-propyl-2-thiouracil (PTU), 6-phenyl-2-thiouracil (PhTU), tapazole (TAP), and 2-mercaptobenzimidazole (MBI) in bovine muscle tissues. Investigation of the retention mechanism of the six analytes on the selected amide-based stationary phase showed that hydrophilic partition was the dominant interaction. The sample preparation included extraction with ACN/H2O (80/20), followed by dispersive solid-phase extraction (d-SPE) with C18 sorbent and hexane partitioning. The method was validated according to European guidelines using internal standards, including isotopically labelled ones. The method's LODs ranged between 2.8 ng g−1 (6-phenyl-2-thiouracil) and 4.1 ng g−1 (2-thiouracil). Application of the proposed method to 48 bovine tissue samples showed non-detectable results. [ABSTRACT FROM AUTHOR]

The assignment of structure by tandem mass spectrometry (MS/MS) relies on the interpretation of the fragmentation behavior of gas‐phase ions. Mass spectra were acquired for a series of heterocyclic mimetics of acidic amino acids and a related series of nitrile amino acids. All amino acids were readily protonated or deprotonated by electrospray ionization (ESI), and distinctive fragmentation processes were observed when the ions were subjected to collision‐induced dissociation (CID). The deprotonated heterocycles showed bond cleavages of the 3‐hydroxyfurazan ring with formation of oxoisocyanate and the complementary deprotonated nitrile amino acid. Further fragmentation of the deprotonated nitrile amino acids was greatly dependent on the length of the alkyl nitrile side chain. Competing losses of CO2 versus HCN occurred from α‐cyanoglycinate (shortest chain), whereas water was lost from 2‐amino‐5‐cyanopentanoate (longest chain). Interestingly, loss of acrylonitrile by a McLafferty‐type fragmentation process was detected for 2‐amino‐4‐cyanobutanoate, and several competing processes were observed for β‐cyanoalanate. In one process, cyanide ion was formed either by consecutive losses of ammonia, carbon dioxide, and acetylene or by a one‐step decarboxylative elimination. In another, complementary ions were obtained from β‐cyanoalanate by loss of acetonitrile or HN=CHCO2H. Fragmentation of the protonated 3‐hydroxyfurazan and nitrile amino acids resulted in the cumulative loss (H2O + CO), a loss that is commonly observed for protonated aliphatic α‐amino acids. Overall, the distinct fragmentation behavior of the multifunctional 3‐hydroxyfurazan amino acids correlated with the charged site, whereas fragmentations of the deprotonated nitrile amino acids showed cooperative interactions between the nitrile and the carboxylate groups. [ABSTRACT FROM AUTHOR]

Τhyreostats (TSs) are veterinary drugs used in livestock farming for fattening. Their administration is banned in the European Union since 1981, and their monitoring for food quality and safety control requires sensitive and confirmatory methods. The present study describes the development and validation of a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6-propyl-2-thiouracil (PTU), 6-phenyl-2-thiouracil (PhTU), tapazole (TAP), and 2-mercaptobenzimidazole (MBI) in bovine muscle tissues. Investigation of the retention mechanism of the six analytes on the selected amide-based stationary phase showed that hydrophilic partition was the dominant interaction. The sample preparation included extraction with ACN/H2O (80/20), followed by dispersive solid-phase extraction (d-SPE) with C18 sorbent and hexane partitioning. The method was validated according to European guidelines using internal standards, including isotopically labelled ones. The method’s LODs ranged between 2.8 ng g−1 (6-phenyl-2-thiouracil) and 4.1 ng g−1 (2-thiouracil). Application of the proposed method to 48 bovine tissue samples showed non-detectable results.

HYDROXAMATE siderophores are low molecular weight, high-affinity ferric iron-chelating compounds which are excreted by Aspergillus fumigatus for iron transport extracellularly or iron storage intracellularly. Aspergillus fumigatus was isolated from different samples, then siderophores were extracted from A. fumigatus by phenol, and diethyl ether, and purified by amberlite XAD-16 resin. The chemical structure of siderophores was described by H¹NMR, ESI-MS/MS, and LC-MS/MS analysis. Also, antimicrobial activity was observed. From the elution profile of siderophores, the major peak was at fraction 28. As a result of H¹NMR analysis, the singlets assigned to the -C=O-CH=C (CH3) C-substructure were detected. The siderophore TafC had antibacterial activity against both G +ve and G -ve bacteria. Under different concentrations of iron, it can be deduced that the concentration of 0.03 mmol of iron at 20 hrs. incubation period was an ideal condition for mycelial fresh weight which positively affected, so production of siderophores increased. From chemical analysis, it was found that the chemical formula C39H58FeN6O15 was consistent with the known siderophore N, N', N"- triacetylfusarinine C (TafC). [ABSTRACT FROM AUTHOR]

Biological bases of autism spectrum disorder (ASD) include both genetic and epigenetic causes. Patients with ASD show anomalies in the profile of certain plasma amino acids, including neuroactive amino acids. Monitoring plasma amino acids may be relevant for patient care and interventions. We evaluated the plasma amino acid profile in samples extracted from dry blood spots by electrospray ionization-tandem mass spectrometry. Fourteen amino acids and eleven amino acid ratios were examined in patients with ASD and intellectual disability (ID), and neurotypical control subjects (TD). The amino acid profile in the ASD group showed reduced levels of ornithine (p = 0.008), phenylalanine (p = 0.042) and tyrosine (p = 0.013). The statistically significant amino acid ratios were Leu+Val/Phe+Tyr (p = 0.002), Tyr/Leu (p = 0.007) and Val/Phe (p = 0.028), such differences remaining significant only in the comparison between ASD and TD. Finally, a positive correlation emerged between the score of the restricted and repetitive behavior on ADOS-2 and the citrulline levels in the ASD group (p = 0.0047). To conclude, patients with ASD may show a distinguishable metabolic profile useful for studying their metabolic pathways in order to develop screening tests and targeted therapies. [ABSTRACT FROM AUTHOR]

• A new method for the online analysis of benzimidazole anthelmintics was developed. • RAMIP-BSA was synthesized and used for online extraction of anthelmintics from milk. • Mebendazole and albendazole were analyzed by ESI-MS/MS without chromatography. • RAMIP-BSA can be used up to 150 times without losing its adsorptive capacity. Multifunctional materials, such as restricted access molecularly imprinted polymers covered with bovine serum albumin (RAMIP-BSA), are effective alternatives for sample preparation techniques. This material selectively adsorbs analytes while excluding macromolecules, enhancing the analysis's efficiency. Among analytical techniques, ESI-MS/MS (Electrospray Ionization-Tandem Mass Spectrometry) has successfully identified and quantified various molecules, including trace-level drugs. Therefore, we proposed, for the first time, an integrated online extraction/analysis system that combines the benefits of RAMIP-BSA and ESI-MS/MS for analyzing mebendazole (MBZ) and albendazole (ABZ) in milk samples without the need for chromatographic separation. Initially, a RAMIP selective for MBZ was synthesized using the bulk method with methacrylic acid and glycidyl methacrylate. Then, the polymer was covered with bovine serum albumin. Subsequently, this adsorbent was packed in a small column and coupled with an ESI-MS/MS instrument in an online configuration. Milli-Q water was used as the loading and reconditioning mobile phases, and a solution of formic acid in methanol (1:100 v/v) was employed as the elution phase. The system enabled simultaneous extraction and determination of MBZ and ABZ in milk samples. The method exhibited linearity between 15.0 and 125.0 μg L−1 for MBZ and 10.0 and 125.0 μg L−1 for ABZ (with a correlation coefficient exceeding 0.99). The limits of quantification were 15.0 and 10.0 μg L−1 for MBZ and ABZ, respectively. Good precision and accuracy were achieved. The developed method was used to analyze MBZ and ABZ in real milk samples and proved to be a viable alternative to conventional sample preparation and chromatographic techniques. [ABSTRACT FROM AUTHOR]

In this study, we have investigated the fragmentation of the widely used steroidal pharmaceutical drugs (n = 14), complexed by a singly charged proton or alkali metal ion (Li+, Na+, K+) using Ion trap and quadrupole time-of-flight mass spectrometers. Spectra were collected by LC-MS/MS analysis using system automated collision energy i.e., of 25–60 eV. Theoretical calculations were also calculated using DFT software. The metal complexes showed different fragmentation pathways not commonly observed for protonated compounds. There was a distinct difference observed in the relative intensities of some common fragments for free vs. metallated drugs. Some major fragments from protonated and lithium adducts showed close resemblance, while sodium and potassium adducts showed different fragments. Theoretical calculations showed a distinct difference in the position of attachment of proton and metals. This adducts ion fragmentation information will be helpful for the identification of these compounds in complex samples.

Tribulus terrestris L. belongs to the family Zygophyllaceae and integral part of various ancient medicinal systems including Chinese, Indian, and European to combat various health ailments. The aim of the present study was to assess the phytochemical constituents, in vitro antioxidant activity using DPPH, FRAP, and H2O2 assays, in vitro anticancer activity using MTT assay, and in vitro and in vivo anti-inflammatory properties of T. terrestris. The acute and sub-acute toxicity of extracts exhibiting most biological potential was examined using murine models. Liquid–liquid partitioning followed by RP–HPLC sub-fraction of crude extract was performed. After that, ESI-MS/MS analysis was done for the timid identification of bioactive metabolites responsible for bioactivities of sub-fractions and HPLC analysis to quantify the compounds using external standards. Among all extracts, T. terrestris methanol extract was noted to hold maximum phenolic (341.3 mg GAE/g) and flavonoid (209 mg QE/g) contents, antioxidant activity in DPPH (IC50 71.4 µg/mL), FRAP (35.3 mmol/g), and H2O2 (65.3% inhibition) assays, anti-inflammatory activities in vitro at 400 µg/mL (heat-induced hemolysis, % inhibition 68.5; egg albumin denaturation, % inhibition 75.6%; serum albumin denaturation, % inhibition 80.2), and in vivo at 200 mg/kg (carrageenan-induced paw edema, % inhibition 69.3%; formaldehyde-induced paw edema, % inhibition 71.3%) and anticancer activity against breast cancer cell (MCF-7) proliferation (IC50 74.1 µg/mL). Acute and sub-acute toxicity studies recorded with no change in body weight, behavior, hematological, serum, and histopathological parameters in treated rats with T. terrestris methanol extracts when compared to control group. Fraction B obtained through liquid–liquid partitioning resulted in more bioactive potential as compared to the parent methanol extract. RP–HPLC analysis of fraction B resulted with four sub-fractions (TBTMF1-TBTMF4), wherein TBTMF3 delineated notable bioactive capabilities as compared to other fractions and parent methanol extract. ESI-MS/MS analysis of TBTMF3 resulted with tentative identification of myricetin, rutin, liquitrigenin, physcion, and protodioscin. It can be stated that T. terrestris is a potential bearing herb and findings of current study further verify the claims made in ancient medicinal systems. However, after investigation of each identified compound, it must be considered for drug discovery. [ABSTRACT FROM AUTHOR]

Lipidomics is a research area that investigates lipidomes that refer to the complete lipid profile within a cell, tissue, organism, or ecosystem by focusing on the interactions with neighboring molecules at the inter- or intracellular spaces of organisms. In recent decades, lipidomics has greatly evolved along with developments in mass spectrometric analysis (i.e., ionization approaches, the types of analyzers, and the data processing tools), leading to a rapid and accurate performance in the identification and quantification of individual lipid species. In this contribution, the identification and quantification of triacylglycerol (TAG) and phospholipid (PL) species in marine sources using mass spectrometry analysis are comprehensively discussed along with their physiological roles and health-beneficial effects on the human body. Moreover, this review emphasizes the importance of the isotopic deconvolution and instrument responses caused by the difference in the carbon number and the unsaturation of TAG and PL species in quantitative analysis. Most TAG and PL detected in marine taxa possess a high proportion of polyunsaturated ω3 long-chain polyunsaturated fatty acids (LCPUFA, ≥C20) in particular eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3), indicating the large number and structural diversity of glycerolipid species containing EPA and DHA. This review also discusses the health effects of TAG and PL species esterified with ω3 LCPUFA. Given that most fisheries science and aquaculture research during recent decades has mainly focused on ω3 LCPUFA after release from parent molecules, discussion of the health effects of ω3 LCPUFA in the form of TAG and PL extends understanding of the bioactivities of ω3 fatty acids as they occur naturally. [ABSTRACT FROM AUTHOR]

Opuntia stricta var. dillenii and Atriplex hortensis var. rubra, two plant species with abundant betalain and flavonoid content, were studied by high performance countercurrent chromatography (HPCCC) coupled with ESI-MS/ MS in an off-line detection process. Both CCC runs used tert.-butyl-methyl-ether/n-butanol/acetonitrile/water 1% trifluoroacetic acid (v/v/v/v, 2:2:1:5) as a biphasic solvent system within an elution-extrusion mode. The resulting fractions were scanned by an off-line sequential injection to ESI-MS/MS approach. The separation of Opuntia stricta var. dillenii extract showed a partial isolation of principal betalains (betanin) from their derivatives (betanidin, phyllocactin, feruloyl-betanin, betaxanthin), degraded products (hydroxyl-neobetanin, mono decarboxy-betanins), and flavonoid glycosides (isorhamnetin-rutinoside, kaempferol-rutinoside). In case of Atriplex hortensis var. rubra, this study confirmed the coexistence of several flavonoids (quercetin-Omalonyl glucoside, kaempferol-O-malonyl glucoside) and generated few pure pigment fractions (celosianin II) for structural elucidation.

Biological bases of autism spectrum disorder (ASD) include both genetic and epigenetic causes. Patients with ASD show anomalies in the profile of certain plasma amino acids, including neuroactive amino acids. Monitoring plasma amino acids may be relevant for patient care and interventions. We evaluated the plasma amino acid profile in samples extracted from dry blood spots by electrospray ionization-tandem mass spectrometry. Fourteen amino acids and eleven amino acid ratios were examined in patients with ASD and intellectual disability (ID), and neurotypical control subjects (TD). The amino acid profile in the ASD group showed reduced levels of ornithine (p = 0.008), phenylalanine (p = 0.042) and tyrosine (p = 0.013). The statistically significant amino acid ratios were Leu+Val/Phe+Tyr (p = 0.002), Tyr/Leu (p = 0.007) and Val/Phe (p = 0.028), such differences remaining significant only in the comparison between ASD and TD. Finally, a positive correlation emerged between the score of the restricted and repetitive behavior on ADOS-2 and the citrulline levels in the ASD group (p = 0.0047). To conclude, patients with ASD may show a distinguishable metabolic profile useful for studying their metabolic pathways in order to develop screening tests and targeted therapies.

Background: Moringa oleifera seed, which is widely utilized as an affordable source of domestic water treatment, is also reputable for its various pharmacological applications globally. Aim: This study evaluates the toxicity and pharmacological potentials of Moringa oleifera seed protein while establishing the chemical profile. Method: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS/MS) were used to analyze the protein/peptide content of the purified protein. The protein was also investigated for in vitro anticancer potential on Hela and MDA-MB-231 cell lines, cytotoxic on 3T3 cell line, and toxicity to brine shrimps. In addition, the antimicrobial action of the protein (polypeptide) was evaluated. Results and Discussion: The MALDI-MS revealed three protein moieties with values of 3.4, 4.6, and 6.9 kDa. On the other hand, LC-ESIMS/ MS analysis revealed dipeptides and tripeptides, which include serinylarginine, leucylproline, leucylmethionyl- glycine, isoleucyl-glycyl-methinine, glycyl-arginyl-aspartic acid, isoleucyl-glutamyl-methionine, diphenylalanine, asparaginyl-aspartyl-histidine, seryl-tyrosyl-tyrosine, phenylalanyl-asparaginyl-tyrosine, and propanolyl-trytophanyl-glycine. The seed protein exhibited extremely low toxicity on both 3T3 and MDA-MB-231 breast cancer cell lines, while the standards, doxorubicin, and cycloheximide had IC50 = 0.5 ± 0.07 and 0.8 ± 0.10 μg/mL, respectively. Further, the protein showed no antibacterial or antifungal activity against all tested organisms. The protein also exhibited no lethality against brine shrimp. The in-silico toxicity results indicated that the peptides are not immunotoxic, carcinogenic, or mutagenic as most belonged to the tox class V except serylarginine predicted for tox class IV (harmful if swallowed). Hence, the intake of the protein remnant in water could be within the tolerable limit. Conclusion: The results obtained suggested that the protein content of the Moringa oleifera seed is non-cytotoxic. The result further validates the safety potential of the defatted and debittered seed material used as potential food sources for both humans and animals. [ABSTRACT FROM AUTHOR]

Tribulus terrestris L. belongs to the family Zygophyllaceae and integral part of various ancient medicinal systems including Chinese, Indian, and European to combat various health ailments. The aim of the present study was to assess the phytochemical constituents, in vitro antioxidant activity using DPPH, FRAP, and H2O2 assays, in vitro anticancer activity using MTT assay, and in vitro and in vivo anti-inflammatory properties of T. terrestris. The acute and sub-acute toxicity of extracts exhibiting most biological potential was examined using murine models. Liquid–liquid partitioning followed by RP–HPLC sub-fraction of crude extract was performed. After that, ESI-MS/MS analysis was done for the timid identification of bioactive metabolites responsible for bioactivities of sub-fractions and HPLC analysis to quantify the compounds using external standards. Among all extracts, T. terrestris methanol extract was noted to hold maximum phenolic (341.3 mg GAE/g) and flavonoid (209 mg QE/g) contents, antioxidant activity in DPPH (IC50 71.4 µg/mL), FRAP (35.3 mmol/g), and H2O2 (65.3% inhibition) assays, anti-inflammatory activities in vitro at 400 µg/mL (heat-induced hemolysis, % inhibition 68.5; egg albumin denaturation, % inhibition 75.6%; serum albumin denaturation, % inhibition 80.2), and in vivo at 200 mg/kg (carrageenan-induced paw edema, % inhibition 69.3%; formaldehyde-induced paw edema, % inhibition 71.3%) and anticancer activity against breast cancer cell (MCF-7) proliferation (IC50 74.1 µg/mL). Acute and sub-acute toxicity studies recorded with no change in body weight, behavior, hematological, serum, and histopathological parameters in treated rats with T. terrestris methanol extracts when compared to control group. Fraction B obtained through liquid–liquid partitioning resulted in more bioactive potential as compared to the parent methanol extract. RP–HPLC analysis of fraction B resulted with four sub-fractions (TBTMF1-TBTMF4), wherein TBTMF3 delineated notable bioactive capabilities as compared to other fractions and parent methanol extract. ESI-MS/MS analysis of TBTMF3 resulted with tentative identification of myricetin, rutin, liquitrigenin, physcion, and protodioscin. It can be stated that T. terrestris is a potential bearing herb and findings of current study further verify the claims made in ancient medicinal systems. However, after investigation of each identified compound, it must be considered for drug discovery.

Non-proteinogenic amino acids are widely explored group of compounds due to their chemical properties and great potential of application in the combinatorial chemistry, medicinal investigation etc. Therefore the synthetic methods of their incorporation to the peptide chain are required. l-Mimosine, (S)-α-amino-β-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid), is a plant amino acid, known to induce apoptosis in human pancreatic cancer xenografts. Here we present our investigations on the synthesis of mimosine-containing peptide and their ESI-MS/MS analysis. We successfully applied Fmoc-protected mimosine a with a free hydroxy ketone group for efficient peptide synthesis in the presence of HATU as a coupling reagent without the formation of side products. Additionally the tandem mass spectrometry analysis revealed the characteristic loss of the heterocyclic ring from mimosine residue side chain. The described method allows insertion of mimosine residue at any endo-position within a peptide sequence. The obtained results may be useful in the synthesis and mass spectrometry analysis of various mimosine-containing peptides. [ABSTRACT FROM AUTHOR]

Depending on the catalyst used, N-methylation of indole with dimethylcarbonate (DMC)--an environmentally friendly alkylation agent--yields different products. With 1,4-diazabicyclo[2.2.2]octane (DABCO), the reaction forms only N-methylated indole, but with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), both N-methylated and N-methoxycarbonylated indole are formed. Using direct ESI(+)-MS monitoring to collect actual snapshots of the changing ionic composition of the reaction solution, we report on the interception and characterization of key intermediates for such reactions. Although a mechanism has been proposed with methoxycarbonylated base as the key intermediate for both DBU and DABCO, the ESI(+)-MS data and B3LYPD3/6-311+G** calculations suggest that the reaction of DMC with indole under either DABCO or DBU catalysis follows contrasting mechanisms. [ABSTRACT FROM AUTHOR]

This manuscript describes the results of a metalloproteomic study of mercury in samples of muscle and liver tissue of the species Serrasalmus rhombeus, popularly known as black piranha and characterised as the most voracious and aggressive predator in the Brazilian Amazon. The metalloproteomic study involved using two-dimensional electrophoresis (2D PAGE) to fractionate the proteome of the muscle and liver tissue samples, along with atomic absorption spectrometry in a graphite furnace (GFAAS) to identify mercury associated with protein SPOTs and mass spectrometry with electrospray ionisation (ESI-MS/MS) to characterise the mercury-binding proteins. The protein SPOTs characterised showed concentrations in the order of 156 mg kg−1, which ranks as the highest concentrations of mercury determined so far in metalloproteomic studies involving fish species in the Amazon region. Based on FASTA sequences of proteins characterised by ESI-MS/MS, bioinformatics studies were performed that allowed identifying nine proteins with characteristics of biomarkers of mercury exposure. Of those proteins, glutathione peroxidase stands out as an enzyme of great importance in the antioxidant defence of organisms subjected to oxidative stress caused by xenobiotics. [ABSTRACT FROM AUTHOR]

Introduction: Black shallot is the novel product of shallot (Allium ascalonicum), one of the most famous spices and remedies used in Vietnamese cuisine and in folk medicine, via aging process in a controlled temperature and humidity environment. However, the bioactivities of black shallot ethanol extract have not been studied yet. Materials & Methods: The aims of this study are determination of chemical composition and antioxidant, anticancer, anti-inflammatory effects of black shallot extract. The chemical constituents of black shallot extract were determined via ESIMS/MS analysis and the antioxidant activity of extract was evaluated by 1,1-diphenyl-2picrylhydrazil (DPPH) radical scavenging assay. The anticancer activity of black shallot against human cervical carcinoma (Hela) and human hepatocellular carcinoma (HepG2) cell lines was tested via the MTT assay. The anti-inflammatory effect of extract was measured through quantification of nitrite oxide production using the Griess reaction. Results & Discussion: We identified the presence of 7 bioactive compounds in black shallot extract, including quercetin 3,4-diglucoside, isorhamnetin 3,4-diglucoside, quercetin 3-glucoside, quercetin 4-glucoside, isorhamnetin 4-glucoside, quercetin aglycone, isrohamnetin. Moreover, black shallot extract exhibited the stronger antioxidant activity than fresh shallot extract (25.67 ± 0.65% versus 18.21 ± 0.35%, respectively). Of note, anticancer and anti-inflammatory effects of black shallot were also higher than those of fresh shallot. Conclusion: The data suggest black shallot as one of the promising functional foods for treatment of inflammatory and cancer related diseases. [ABSTRACT FROM AUTHOR]

Glutelins derived from oats were purified and hydrolyzed sequentially using alcalase proteases for production of antioxidant peptides. The antioxidant activities of oat glutelin peptides were evaluated using reducing power methods including hydroxyl and 1,1-diphenyl-2-pycrylhydrazyl (DPPH) radical-scavenging assays. The results demonstrated that oats glutelin peptide D-1a exhibited the highest antioxidant activity compared to the other hydrolysates. D-1a was identified to be His–Tyr–Asn–Ala–Pro–Ala–Leu (784.38 Da) by electrospray ionization mass spectrometry (ESI-MS/MS).

A simple, rapid and selective high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for simultaneous determination of three withanolides (withaferin A, withanone and withanolide A) and three phenolic acids (caffeic acid, ferulic acid and benzoic acid) from different parts (root, stem and leaf) of Withania somnifera and its two commercially available polyherbal formulations. The extraction efficiency of withanolides and phenolic acids were tested using two solvents, chloroform and methanol, respectively. HPTLC separation was performed on silica coated aluminium plates Si 60F 254 ; using toluene, ethyl acetate and acetic acid (60:40:4). The samples were quantitated at 231 nm. The purity and identity of peaks of all the six analytes were confirmed by matching Rf values and UV-spectrum with authentic standards. The identity of three withanolides was further confirmed by positive ion electrospray ionization mass spectrometry (ESI-MS/MS) analyses. The developed method was validated for sensitivity, linearity, reproducibility, accuracy, the limit of detection (LOD) and limit of quantification (LOQ) following the guidelines of the International Conference on Harmonization (ICH). The method was found to be linear (r > 0.99) in the range of 50–2000 ng/band for benzoic acid and 50–1000 ng/band for the other five studied metabolites. This simple and accurate HPTLC method provided enhanced resolution of studied analytes as compared to other phytoconstituents present in W. somnifera extracts. It has also been successfully applied in the analysis and quantification of two polyherbal formulations containing W. somnifera plant parts. • HPTLC method was developed for specific separation of withanolides and phenolic acids from Withania somnifera plant extracts. • Six analytes (withaferin A, withanone, withanolide A, caffeic, ferulic-, and benzoic acids) can be determined by this method. • The method was validated according to ICH guidelines. • This cost-effective method can be used for quantifying specific withanolides and phenolic acids from herbal products. [ABSTRACT FROM AUTHOR]

Natural 2H‐chromenes were isolated from the crude extract of Piper aduncum (Piperaceae) and analyzed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) applying collision‐induced dissociation. Density functional theory (DFT) calculations were used to explain the preferred protonation sites of the 2H‐chromenes based on thermochemical parameters, including atomic charges, proton affinity, and gas‐phase basicity. After identifying the nucleophilic sites, the pathways were proposed to justify the formation of the diagnostic ions under ESI‐MS/MS conditions. The calculated relative energy for each pathway was in good agreement with the energy‐resolved plot obtained from ESI‐MS/MS data. Moreover, the 2H‐chromene underwent proton attachment on the prenyl moiety via a six‐membered transition state. This behavior resulted in the formation of a diagnostic ion due to 2‐methylpropene loss. These studies provide novel insights into gas‐phase dissociation for natural benzopyran compounds, indicating how reactivity is correlated to the intrinsic acid‐base equilibrium and structural aspects, including the substitution pattern on the aromatic moiety. Therefore, these results can be applied in the identification of benzopyran derivatives in a variety of biological samples. [ABSTRACT FROM AUTHOR]

Graphical abstract Differentation of open-cage fullerene isomers by dissociation of sodiated monomers (using LIFT) and dimers (using CID) reveals multiple interaction motives. Highlights • Ion formation of open-cage fullerenes (OCFs) by DCTB- / pencil lead- MALDI and ESI. • MS2 spectra of the molecular ions of isomeric OCFs are indistinguishable. • MS2 spectra of sodiated and potassiated OCFs differentiate between isomers. • Metal coordination to the heteroatoms at the cage orifice causes differentiation. • Relative sodium ion affinities are obtained by the kinetic method. Abstract Isomeric open-cage fullerenes (OCFs) are studied by MALDI-LIFT. The two types of isomers feature different connectivities of the heteroatoms at the rim of the orifice to the fullerene cage. The molecular ions (radical cations, M+.) are generated by electron transfer MALDI, using DCTB as the matrix. The resulting LIFT spectra are indistinguishable. The alternative use of pencil lead is found inferior as a matrix but proves an exceptionally facile method of creating sodium and potassium adducts. The fragmentation of these metallated OCFs reveals different fragmentation pathways allowing the differentiation of the OCF isomers. This is evidently caused by the different interactions of the metal cation with the heteroatoms at the rim of the orifice. Additionally, the influence of the functionalization and isomer structure on the sodium ion affinity (SIA) is analysed by the kinetic method investigating the dissociations of sodium-bound hetero-dimers in electrospray ionization/ collision-induced dissociation (ESI-CID) experiments. [ABSTRACT FROM AUTHOR]

Highlights • Peach phenolics were separated and identified by Sephadex LH-20 chromatography and HPLC-ESI-MS. • New phenolics found included an eriodictyol derivative and quercetin diglycoside. • The high-molecular-weight phenolic fraction is a major contributor to the antioxidant capacity. • Significant differences in phenolic contents were noted among the six Georgia cultivars examined. Abstract Acetonic crude phenolic extracts of six Georgia peach cultivars were prepared and separated into low- and high-molecular-weight (LMW and HMW) fractions by Sephadex LH-20 column chromatography. Further characterization via RP-HPLC-ESI-MS identified the main phenolics as hydroxycinnamates, (+)-catechin, and proanthocyanidins with degrees of polymerization up to seven. The LMW phenolics of the commercial cultivar, ‘July Prince’, were further chromatographed and examined by RP-HPLC-ESI-MS. Derivatives of phenolic acids and flavan-3-ols, along with eriodictyol and quercetin diglycosides, were identified. Antioxidant capacities of the LMW and HMW fractions were determined using in vitro assays. H-ORAC FL and FRAP assays gave values of 872 to 2428 μmol Trolox eq./100 g f.w. and 309 to 432 μmol Fe2+ eq./100 g f.w., respectively. The total phenolics content (TPC) was also measured; correlations between TPCs and antioxidant assays indicated that the HMW fractions of peach extracts were major contributors to the antioxidant capacity of the cultivars analyzed. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is currently diagnosed according to behavioral criteria. Biomarkers that identify children with ASD could lead to more accurate and early diagnosis. ASD is a complex disorder with multifactorial and heterogeneous etiology supporting recognition of biomarkers that identify patient subsets. We investigated an easily testable blood metabolic profile associated with ASD diagnosis using high throughput analyses of samples extracted from dried blood spots (DBS). A targeted panel of 45 ASD analytes including acyl-carnitines and amino acids extracted from DBS was examined in 83 children with ASD (60 males; age 6.06 ± 3.58, range: 2–10 years) and 79 matched, neurotypical (NT) control children (57 males; age 6.8 ± 4.11 years, range 2.5–11 years). Based on their chronological ages, participants were divided in two groups: younger or older than 5 years. Two-sided T-tests were used to identify significant differences in measured metabolite levels between groups. Näive Bayes algorithm trained on the identified metabolites was used to profile children with ASD vs. NT controls. Of the 45 analyzed metabolites, nine (20%) were significantly increased in ASD patients including the amino acid citrulline and acyl-carnitines C2, C4DC/C5OH, C10, C12, C14:2, C16, C16:1, C18:1 (P: < 0.001). Näive Bayes algorithm using acyl-carnitine metabolites which were identified as significantly abnormal showed the highest performances for classifying ASD in children younger than 5 years (n: 42; mean age 3.26 ± 0.89) with 72.3% sensitivity (95% CI: 71.3;73.9), 72.1% specificity (95% CI: 71.2;72.9) and a diagnostic odds ratio 11.25 (95% CI: 9.47;17.7). Re-test analyses as a measure of validity showed an accuracy of 73% in children with ASD aged ≤ 5 years. This easily testable, non-invasive profile in DBS may support recognition of metabolic ASD individuals aged ≤ 5 years and represents a potential complementary tool to improve diagnosis at earlier stages of ASD development.

Outer membrane vesicles (OMVs) of gram-negative bacteria are released during all growth phases and play an important role in bacterial physiology. They consist of lipids, proteins, lipopolysaccharides and other molecules. The OMVs of the Antarctic bacterium Pseudomonas syringae Lz 4W were isolated and identified their proteins. The mass spectral data set deposited with PRIDE, accession number PXD 000221 is presented in this report. The proteins identified from the OMVs of P. syringae Lz4W, data of this study were published in the Journal of proteome research [1].

Consumers’ interest in the way meat is produced is increasing in Europe. The resulting free range and organic meat products retail at a higher price, but are difficult to differentiate from their counterparts. To ascertain authenticity and prevent fraud, relevant markers need to be identified and new analytical methodology developed. The objective of this pilot study was to characterize pork belly meats of different animal welfare classes by their fatty acid (Fatty Acid Methyl Ester—FAME), non-volatile compound (electrospray ionization-tandem mass spectrometry—ESI-MS/MS), and volatile compound (proton-transfer-reaction mass spectrometry—PTR-MS) fingerprints. Well-defined pork belly meat samples (13 conventional, 15 free range, and 13 organic) originating from the Netherlands were subjected to analysis. Fingerprints appeared to be specific for the three categories, and resulted in 100%, 95.3%, and 95.3% correct identity predictions of training set samples for FAME, ESI-MS/MS, and PTR-MS respectively and slightly lower scores for the validation set. Organic meat was also well discriminated from the other two categories with 100% success rates for the training set for all three analytical approaches. Ten out of 25 FAs showed significant differences in abundance between organic meat and the other categories, free range meat differed significantly for 6 out of the 25 FAs. Overall, FAME fingerprinting presented highest discrimination power.

Wax esters (WEs) and steryl esters (SEs) are minor components of sunflower oils formed by the esterification of long chain fatty alcohols and sterols to fatty acids. These compounds have similar carbon numbers and polarities making them difficult to separate using conventional chromatographic methods. In this study, electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) allowed the rapid and accurate profiling of WEs and SEs acyl moieties in total ester fractions of common and mutant sunflower oils with different fatty acid profiles. The acyl composition of both WEs and SEs partially reflected that of the oil and the high oleic background displayed the lowest level of crystallisable waxes. ESI-MS/MS complemented by GC–MS analyses revealed that SEs contain 17–30% of previously unreported methylsterol moieties. We demonstrated that these compounds are overlooked by official sterol analytical methods which may have consequences for quality control and authentication of vegetable oils prior to commercialisation. [ABSTRACT FROM AUTHOR]

This study presents data on the extraction and characterization of proteins associated with mercury in the muscle and liver tissues of jaraqui (Semaprochilodus spp.) from the Madeira River in the Brazilian Amazon. Protein fractionation was carried out by two-dimensional electrophoresis (2D-PAGE). Mercury determination in tissues, pellets, and protein spots was performed by graphite furnace atomic absorption spectrometry (GFAAS). Proteins in the spots that showed mercury were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The highest mercury concentrations were found in liver tissues and pellets (426 ± 6 and 277 ± 4 μg kg−1), followed by muscle tissues and pellets (132 ± 4 and 86 ± 1 μg kg−1, respectively). Mercury quantification in the protein spots allowed us to propose stoichiometric ratios in the range of 1-4 mercury atoms per molecule of protein in the protein spots. The proteins characterized in the analysis by ESI-MS/MS were keratin, type II cytoskeletal 8, parvalbumin beta, parvalbumin-2, ubiquitin-40S ribosomal S27a, 39S ribosomal protein L36 mitochondrial, hemoglobin subunit beta, and hemoglobin subunit beta-A/B. The results suggest that proteins such as ubiquitin-40S ribosomal protein S27a, which have specific domains, possibly zinc finger, can be used as biomarkers of mercury, whereas mercury and zinc present characteristics of soft acids. [ABSTRACT FROM AUTHOR]

Low molecular weight heparins (LMWHs) are widely used anticoagulant drugs. The composition and sequence of LMWH oligosaccharides determine their safety and efficacy. The short oligosaccharide pool in LMWHs undergoes more depolymerization reactions than the longer chains and is the most sensitive indicator of the manufacturing process. Electrospray ionization tandem mass spectrometry (ESI–MS/MS) has been demonstrated as a powerful tool to sequence synthetic heparin oligosaccharide but never been applied to analyze complicated mixture like LMWHs. We established an offline strong anion exchange (SAX)-high performance liquid chromatography (HPLC) and ESI–MS/MS approach to sequence the short oligosaccharides of dalteparin sodium. With the help of in-house developed MS/MS interpretation software, the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained. Interestingly, we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC–MS/MS analysis. This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization. [ABSTRACT FROM AUTHOR]

Psidium guajava L. have been reported to be a rich source of antioxidant compounds. Its carotenoids have been highlighted by their high antioxidant capacity, which offers several benefits for human health. In this sense, lycopene isomers need to be identified. In this work, the comprehensive chemical characterization, by HPLC‐DAD, MS/MS, and NMR, of lycopene isolated from P. guajava L., antioxidant and antimicrobial activity of lycopene extracts and isolated lycopene are evaluated. The FTIR results reported a structure with Z configuration, confirmed by UV‐Vis, with λmax = 448, 473, and 505 nm for 5‐Z lycopene. Furthermore, MS/MS positive ionization shows one fragment m/z 309 [M‐227]+, relatively abundant for isolated lycopene. Experimental and Theoretical NMR studies revealed that guava may contain 5‐Z lycopene because of the similarity found among the peaks. Lycopene extracts presented higher antioxidant activity than isolated lycopene, from both P. guajava L. and tomato, when measured by ABTS and ORAC (r2 = 0.9995 and r2 = 0.9992, respectively). In addition, lycopene extract shows antibacterial efficacy against E. coli, S. aureus, and L. innocua, presenting MBC values of 20 mg mL−1. These results suggest that lycopene extract have potential applicability for food, cosmetics, and pharmaceutical industry. Practical Applications: Lycopene from P. guajava L. is characterized by HPLC‐DAD, MS, NMR, FTIR, and X‐Ray, presented antioxidant capacity by ORAC and antibacterial efficacy against food pathogens. Lycopene is isolated from P. guajava L., and lycopene extracts produced are characterized by HPLC‐DAD and ESI‐MS/MS. Lycopene isolated from P. guajava L. following the process may be 5‐Z isomer of lycopene. [ABSTRACT FROM AUTHOR]

Glutelins derived from oats were purified and hydrolyzed sequentially using alcalase proteases for production of antioxidant peptides. The antioxidant activities of oat glutelin peptides were evaluated using reducing power methods including hydroxyl and 1,1-diphenyl-2-pycrylhydrazyl (DPPH) radical-scavenging assays. The results demonstrated that oats glutelin peptide D-1a exhibited the highest antioxidant activity compared to the other hydrolysates. D-1a was identified to be His–Tyr–Asn–Ala–Pro–Ala–Leu (784.38 Da) by electrospray ionization mass spectrometry (ESI-MS/MS). [ABSTRACT FROM AUTHOR]

In this study, mannanoligosaccharides (MOS) were isolated from palm kernel cake by aqueous extraction using high temperature and pressure. Structural characterization of MOS was carried out using acid hydrolysis, methylation analysis, ESI-MS/MS and 1D/2D NMR. The prebiotic activity of MOS was evaluated in vitro using two probiotic Lactobacillus strains. Sugar analysis indicated the presence of mannose in each of the oligomers. Methylation and 1D/2D NMR analysis indicated that the MOS have a linear structure consisting of (1→4)- β - d -mannopyranosyl residues. ESI-MS/MS results showed that the isolated mannan oligomers, MOS-III, MOS-IV, MOS-V and MOS-VI consist of tetra–, penta–, hexa–, and hepta–saccharides with molecular weights of 689, 851, 1013 and 1151 Da, respectively. Based on the in vitro growth study, MOS-III and MOS-IV was found to be effective in selectively promoting the growth of Lactobacillus reuteri C1 strain as evidenced by the optical density of the culture broth. [ABSTRACT FROM AUTHOR]

Antioxidant and angiotensin converting enzyme ( ACE) inhibitory peptides were identified in a 5 kDa ultrafiltration permeate of a whey protein hydrolysate generated at semi-pilot scale. Further laboratory scale ultrafiltration of this 5 kDa permeate resulted in a 0.65 kDa permeate with antioxidant, (1.11 ± 0.074 μmol TE per mg dry weight, oxygen radical absorbance capacity, ORAC) and ACE inhibitory ( ACE IC50 0.215 ± 0.043 mg mL−1) activities. Semi-preparative ( SP) reverse phase high-performance liquid chromatography ( RP- HPLC) of the 0.65 kDa permeate resulted in a fraction ( SP_F3) with a 4.4-fold increase in ORAC activity (4.83 ± 0.45 μmol TE mg dry weight) and a 1.3-fold increase in ACE inhibitory activity (84.35 ± 1.36% inhibition when assayed at 0.28 mg mL−1). Peptides within SP_F3 were identified using UPLC- ESI- MS/ MS. Met-Pro-Ile had the highest ORAC activity (205.75 ± 12.08 μmol TE per mmol peptide) while Met-Ala-Ala and Val-Ala-Gly-Thr had the highest ACE inhibitory activities ( IC50:515.50 ± 1.11 and 610.30 ± 2.41 μ m, respectively). [ABSTRACT FROM AUTHOR]

An analytical method using liquid chromatography-tandem mass spectrometry has been developed for the determination of 5 paralytic shellfish poisoning (PSP) toxins (saxitoxin, gonyautoxin1-4) in shellfish and tunicate. The tested parameters included extraction solvent, clean-up method, mobile phase and mass spectrometric detection parameters including multiple reaction monitoring (MRM) conditions. The PSP toxins were extracted with 0.1 N hydrochloric acid and cleaned up using C18 SPE cartridges. Chromatographic separation of the 5 PSP toxins was carried out on a TSK-gel Amide-80 column with gradient elution. The developed method was validated according to the requirements of SANCO 12571/2013. The average recoveries of the PSP toxins ranged from 82.7% to 110.7%, the intra-day precisions were in the range of 0.3–10.9%, the inter-day precisions were in the range of 0.8–10.0% and the limits of quantification (LOQs) were in the range of 25–100 μg/kg, depending on the analytes and matrices. The validated method was successfully applied for the determination of 5 PSP toxins in real shellfish and tunicate samples, including mussel, oyster, ark shell, sea squirt and styela clava. [ABSTRACT FROM AUTHOR]

Bile acid synthesis defects are rare genetic disorders characterized by a failure to produce normal bile acids (BAs), and by an accumulation of unusual and intermediary cholanoids. Measurements of cholanoids in urine samples by mass spectrometry are a gold standard for the diagnosis of these diseases. In this work improved methods for the chemical synthesis of 30 BAs conjugated with glycine, taurine and sulfate were developed. Diethyl phosphorocyanidate (DEPC) and diphenyl phosphoryl azide (DPPA) were used as coupling reagents for glycine and taurine conjugation. Sulfated BAs were obtained by sulfur trioxide-triethylamine complex (SO 3 -TEA) as sulfating agent and thereafter conjugated with glycine and taurine. All products were characterized by NMR, IR spectroscopy and high resolution mass spectrometry (HRMS). The use of these compounds as internal standards allows an improved accuracy of both identification and quantification of urinary bile acids. [ABSTRACT FROM AUTHOR]

A comparative panoramic mass spectrometric analysis of serum samples of children suffering from autistic disorders was carried out. Three families were examined, with nine samples distributed in four control samples of healthy parents and five samples of autistic children. In the course of comparative analysis of protein composition of the serum samples, a small group of potential marker proteins for diagnosis was selected, which are not functionally or structurally related to one another. The group included 15 proteins; one of them, LIM domain-containing protein 1, was identified in four out of the five autism depleted serum samples, and the other 14 were identified in three samples. This could be the basis to develop highly sensitive quantitative mass spectrometric SRM methods to detect autism markers in serum. [ABSTRACT FROM AUTHOR]

Imperata cylindrica (L.) Raeusch. (IMP) aerial part ethyl acetate extract has anti-proliferative, pro-apoptotic, and pro-oxidative effects towards colorectal cancer in vitro. The chemical constituents of IMP aerial part ethyl acetate extract were isolated using high-performance liquid chromatography (HPLC) and identified with tandem mass spectrometry (ESI-MS/MS) in combination with ultraviolet-visible spectrophotometry and 400 MHz NMR. The growth inhibitory effects of each identified component on BT-549 (breast) and HT-29 (colon) cancer cell lines were evaluated after 48/72 h treatment by MTT assay. Four isolated compounds were identified as trans-p-Coumaric acid (1); 2-Methoxyestrone (2); 11, 16-Dihydroxypregn-4-ene-3, 20-dione (3); and Tricin (4). Compounds (2), (3), and (4) exhibited considerable growth inhibitory activities against BT-549 and HT-29 cancer cell lines. Compounds (2), (3), and (4) are potential candidates for novel anti-cancer agents against breast and colorectal cancers.

A rapid, precise, specific, and accurate Electrospray Ionisation Tandem Mass Spectrometry (ESI-MS / MS) method has been developed and subsequently validated, for the determination of pregabalin (PB) in human plasma. Gabapentin (GB) was used as the internal standard. PB and GB were extracted from the plasma using a combination of deproteinization, using 0.1% formic acid and liquid-liquid extraction, using methylene chloride. PB and GB were separated using the Hypurity advance column (50 mm × 4.6 mm, 5 μm) and mobile phase, consisting of methanol : 0.1% formic acid (80:20 v / v). PB was determined by using ESI-MS / MS in positive ion mode, with the help of the API 2000 spectrophotometer, operated in a multiple reaction monitoring mode. The parent-to-product ion combination of m / z 160.2→55.1 and 172.2→95.0 was used to quantify PB and GB, respectively. The assay was validated in the concentration range of 99.79 - 4019.90 ng / mL for PB. The limit of quantification (LOQ) was identifiable and reproducible at 99.79 ng / mL. The method has been successfully applied to study the pharmacokinetics of PB in healthy male volunteers.

In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the diversity of endophytic bacteria from ethnovarieties of cassava cultivated by Brazilian Amazon Indian tribes and also to study the secondary metabolites produced by a Bacillus pumilus strain. Sixty seven cassava endophytic bacteria were subjected to 16S rRNA sequencing and FAME analysis. The bacterial profile revealed that 25% of all endophytic isolates belonged to the genus Bacillus. The isolate B. pumilus MAIIIM4a showed a strong inhibitory activity against the fungi Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii. Secondary metabolites of this strain were extracted using hexane, dichloromethane and ethyl acetate. Extracts were subjected to bioautography and LC/MS analysis, which allowed the identification of pumilacidin, an antifungal compound produced by B. pumilus MAIIIM4a. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy.Na busca de novos organismos e novos metabólitos secundários, um estudo foi conduzido visando avaliar a diversidade de bactérias endofíticas de etnovariedades de mandioca cultivadas por tribos indígenas da Amazônia brasileira e também para estudar metabólitos secundários produzidos por Bacillus pumilus. Sessenta e sete bactérias endofíticas de mandioca foram identificadas através do seqüenciamento do gene 16S rRNA e por meio da análise de ácidos graxos (FAME). Essas análises revelaram que 25% de todos os endofíticos pertenciam ao gênero Bacillus. O isolado Bacillus pumilus MAIIIM4a apresentou forte ação inibitória contra os fitopatógenos Rhizoctonia solani, Pythium aphanidermatum e Sclerotium rolfsii. Os metabólitos secundários deste isolado foram extraídos do sobrenadante usando-se hexano, diclorometano e acetato de etila. Esses extratos foram utilizados nas análises de bioautografia e LC-MS, as quais permitiram a identificação do composto pumilacidina, um antifúngico produzido por B. pumilus MAIIIM4a. A localização das bactérias endofíticas foi confirmada examinando-se o tecido celular da mandioca através de microscopia eletrônica.

Polycationic carbohydrates represent an attractive class of biomolecules for several applications and particularly as non viral gene delivery vectors. In this case, the establishment of structure-biological activity relationship requires sensitive and accurate characterization tools to both control and achieve fine structural deciphering. Electrospray-tandem mass spectrometry (ESI-MS/MS) appears as a suitable approach to address these questions. In the study herein, we have investigated the usefulness of electron transfer dissociation (ETD) to get structural data about five polycationic carbohydrates demonstrated as promising gene delivery agents. A particular attention was paid to determine the influence of charge states as well as both fluoranthene reaction time and supplementary activation (SA) on production of charge reduced species, fragmentation yield, varying from 2 to 62%, as well as to obtain the most higher both diversity and intensity of fragments, according to charge states and targeted compounds. ETD fragmentation appeared to be mainly directed toward pending group rather than carbohydrate cyclic scaffold leading to a partial sequencing for building blocks when amino groups are close to carbohydrate core, but allowing to complete structural deciphering of some of them, such as those including dithioureidocysteaminyl group which was not possible with CID only. Such findings clearly highlight the potential to help the rational choice of the suitable analytical conditions, according to the nature of the gene delivery molecules exhibiting polycationic features. Moreover, our ETD-MS/MS approach open the way to a fine sequencing/identification of grafted groups carried on various sets of oligo-/polysaccharides in various fields such as glycobiology or nanomaterials, even with unknown or questionable extraction, synthesis or modification steps. [ABSTRACT FROM AUTHOR]

The visceral mass of the gastropod, Harpa ventricosa was hydrolysed using trypsin, alcalase and pepsin for 12 h to produce protein hydrolysates. Subsequently, the active hydrolysate was observed in the 3rd hour of tryptic hydrolysate (29.17 ± 0.62 and 34.85 ± 0.55 %) using human red blood cell (HRBC) membrane stabilization and albumin denaturation (AD) assays. The active hydrolysate was fractionated by membrane filtration unit, where <10-kDa fraction revealed better anti-inflammatory activity with IC value 6.27 ± 0.05 and 5.38 ± 0.02 mg/ml for HRBC and AD assays, respectively. Additionally, the active fraction contains essential and non-essential (aspartic acid, arginine, glutamic acid and leucine) amino acids and, sequentially, the active fraction was further purified using consecutive chromatography, in which fraction C-II exhibited strong anti-inflammatory activity (HRBC 56.02 ± 0.52 and AD 50.71 ± 1.10 % assays). The non-toxic, low molecular weight (690.2 Da) hexapeptide (Ala-Lys-Gly-Thr-Trp-Lys) suppressed the nitric oxide (NO) and pro-cytokine production in a dose-dependent manner on THP-1 cell lines. [ABSTRACT FROM AUTHOR]

This study was aimed to assess the effect of time and temperature on the extraction of antioxidant compounds from jabuticaba seeds ( Myrciaria cauliflora cv. Sabará), to optimize the solvent proportion (water, ethyl alcohol, and propanone), and to characterize the extract according to the chemical composition, antioxidant, and antimicrobial properties. Proximal composition, total phenolic content (TPC), antioxidant, and antimicrobial activities were analyzed. The optimized solvent ratio of 60% water and 40% propanone provided a mean TPC of 8.65 g GAE/100 g seeds and the antioxidant activity toward 2,2-diphenyl-1-picrylhydrazyl (DPPH) was 82.79% ± 0.50%. Time and temperature parameters did not influence the yield of TPC. The gross seed extract was partially purified and both exhibited a high antioxidant activity and antimicrobial potential toward Gram-positive and Gram-negative bacteria. The purified jabuticaba seed lyophilized extract contained a higher ( P < 0.05) TPC, o-diphenols, flavonols, and antioxidant activity measured by the DPPH assay and total reducing capacity as compared to the gross lyophilized extract. Electrospray ionization coupled with tandem mass spectrometry (ESI-MS/MS) data showed the presence of ellagitannins and ellagic acid in the extracts, which are probably the responsible for the antimicrobial and antioxidant activities. [ABSTRACT FROM AUTHOR]