Charge site manipulation to enhance top-down fragmentation efficiency.

In recent years, top-down mass spectrometry has become a widely used approach to study proteoforms; however, improving sequence coverage remains an important goal. Here, two different proteins, α-synuclein and bovine carbonic anhydrase, were subjected to top-down collision-induced dissociation (CID) after electrospray ionisation. Two high-boiling solvents, DMSO and propylene carbonate, were added to the protein solution in low concentration (2%) and the effects on the top-down fragmentation patterns of the proteins were systematically investigated. Each sample was measured in triplicate, which revealed highly reproducible differences in the top-down CID fragmentation patterns in the presence of a solution additive, even if the same precursor charge state was isolated in the quadrupole of the instrument. Further investigation supports the solution condition-dependent selective formation of different protonation site isomers as the underlying cause of these differences. Higher sequence coverage was often observed in the presence of additives, and the benefits of this approach became even more evident when datasets from different solution conditions were combined, as increases up to 35% in cleavage coverage were obtained. Overall, this approach therefore represents a promising opportunity to increase top-down fragmentation efficiency.

Thirty years of fruitful collaborations between a physician and mass spectrometrists in diabetes field.

The nonenzymatic protein glycation and the subsequent formation of advanced glycation end products is a process involved in the long-term complications of diabetes. In this context the collaboration, in the last 30 years, between my research group, operating in the DPT of Medicine of Padua University, and the mass spectrometric group, operating in CNR of Padua, are described and discussed. The development of new mass spectrometric techniques has allowed investigation more indepth, starting from the applications on small molecules responsible for the browning observed in the interactions between sugars and proteins, and growing up to intact proteins as albumin, immunoglobulin, hemoglobin, and so forth, with the determination of their glycation levels as well as their glycation sites. This study has helped to clarify the role of advanced glycation end products in the pathogenesis of the chronic complications of diabetes. In particular the results obtained in diabetic nephropathy, diabetic cardiovascular disease and in placenta samples of patients affected by gestational diabetes are described in this review.

Suppressing the background of LC-ESI-MS analysis of permethylated glycans using the active background ion reduction device.

Mass spectrometry (MS) has revolutionized analytical chemistry, enabling precise identification and quantification of chemical species, which is pivotal for biomarker discovery and understanding complex biological systems. Despite its versatility, the presence of background ions in MS analysis hinders the sensitive detection of low-abundance analytes. Therefore, studies aimed at lowering background ion levels have become increasingly important. Here, we utilized the commercially available Active Background Ion Reduction Device (ABIRD) to suppress background ions and assess its effect on the liquid chromatography-electrospray ionization (LC-ESI)-MS analyses of N-glycans on the Q Exactive HF mass spectrometer. We also investigated the effect of different solvent vapors in the ESI source on N-glycan analysis by MS. ABIRD generally had no effect on high-mannose and neutral structures but reduced the intensity of some structures that contained sialic acid, fucose, or both when methanol vapor filled the ESI source. Based on our findings on the highest number of identified N-glycans from human serum, methanol vapor in the ion source compartment may enhance N-glycan LC-ESI-MS analyses by improving the desolvation of droplets formed during the ESI process due to its high volatility. This protocol may be further validated and extended to advanced bottom-up proteomic/glycoproteomic studies for the analysis of peptide/glycopeptide ions by MS.

Mn(II) Promoted Divergent-Convergent Domino Reaction Giving Dinuclear Tetrasubstituted Pyrrole Complex.

Domino reaction of benzo[d]thiazole-2-methylamine (S1) has been developed in the presence of MnCl2 ⋅ 4H2O, leading to tetrasubstituted pyrrole coordinated dinuclear Mn(II) complex 1 ([MnClP]2, P-=2,3,4,5-tetrakis(benzo[d]thiazol-2-yl)pyrrol-1-ide). The reaction process has been studied by assigning a series of intermediates based on time-dependent mass spectrometry, control experiments, crystallography, and density functional theory (DFT) theoretical calculation. A plausible mechanism involving an unprecedented divergent-convergent domino sequence has been proposed. Compound S1 could be activated by MnCl2 ⋅ 4H2O via coordination, which divergently produces two intermediates imine II (1-(benzo[d]thiazol-2-yl)-N-(benzo[d]thiazol-2-ylmethyl)methanimine) and alkene C (1,2-bis(benzo[d]thiazol-2-yl)ethene) through oxidative self-condensation and free radical coupling followed by elimination, respectively. They could then react with each other convergently via formal [3+2] cycloaddition to give deprotonated tetrasubstituted pyrrole coordinated intermediate [MnClP] after aromatization. Dimerization of [MnClP] produces the final product 1. Three C-C bonds and one C-N bond are formed through this six-step domino sequence. The corresponding organic skeleton (HP: 2,2',2'',2'''-(1H-pyrrole-2,3,4,5-tetrayl)tetrakis(benzo[d]thiazole)) has been obtained from 1 and shows a higher fluorescent quantum yield (52 %) than the reported 3,4-diphenyl substituted analogue 2,2'-(3,4-diphenyl-1H-pyrrole-2,5-diyl)bis(benzo[d]thiazole) (DPB) (42 %).

Protein-Protein Stabilization in VIVO/8-Hydroxyquinoline-Lysozyme Adducts.

The binding of the potential drug [VIVO(8-HQ)2], where 8-HQ is 8-hydroxyquinolinato, with hen egg white lysozyme (HEWL) was evaluated through spectroscopic (electron paramagnetic resonance, EPR, and UV-visible), spectrometric (electrospray ionization-mass spectrometry, ESI-MS), crystallographic (X-ray diffraction, XRD), and computational (DFT and docking) studies. ESI-MS indicates the interaction of [VIVO(8-HQ)(H2O)]+ and [VIVO(8-HQ)2(H2O)] species with HEWL. Room temperature EPR spectra suggest both covalent and non-covalent binding of the two different V-containing fragments. XRD analyses confirm these findings, showing that [VIVO(8-HQ)(H2O)]+ interacts covalently with the solvent exposed Asp119, while cis-[VIVO(8-HQ)2(H2O)] non-covalently with Arg128 and Lys96 from a symmetry mate. The covalent binding of [VIVO(8-HQ)(H2O)]+ to Asp119 is favored by a π-π contact with Trp62 and a H-bond with Asn103 of a symmetry-related molecule. Additionally, the covalent binding of VVO2 + to Asp48 and non-covalent binding of other V-containing fragments to Arg5, Cys6, and Glu7 are revealed. Molecular docking indicates that, in the absence of the interactions occurring at the protein-protein interface close to Asp119, the covalent binding to Glu35 or Asp52 should be preferred. Such a protein-protein stabilization could be more common than what believed up today, at least in the solid state, and should be considered in the characterization of metal-protein adducts.

Extraction of naturally occurring peptides versus the tryptic digestion of proteins from fetal versus adult bovine serum for LC-ESI-MS/MS.

The naturally occurring peptides and digested proteins of fetal versus adult bovine serum were compared by LC-ESI-MS/MS after correction against noise from blank injections and random MS/MS spectra as statistical controls. Serum peptides were extracted by differential precipitation with mixtures of acetonitrile and water. Serum proteins were separated by partition chromatography over quaternary amine resin followed by tryptic digestion. The rigorous X!TANDEM goodness of fit algorithm that has a low error rate as demonstrated by low FDR q-values (q ≤ 0.01) showed qualitative and quantitative agreement with the SEQUEST cross correlation algorithm on 12,052 protein gene symbols. Tryptic digestion provided a quantitative identification of the serum proteins where observation frequency reflected known high abundance. In contrast, the naturally occurring peptides reflected the cleavage of common serum proteins such as C4A, C3, FGB, HPX, A2M but also proteins in lower concentration such as F13A1, IK, collagens and protocadherins. Proteins associated with cellular growth and development such as actins (ACT), ribosomal proteins like Ribosomal protein S6 (RPS6), synthetic enzymes and extracellular matrix factors were enriched in fetal calf serum. In contrast to the large literature from cord blood, IgG light chains were absent from fetal serum as observed by LC-ESI-MS/MS and confirmed by ELISA.

Sheet Models for Methylaluminoxane (MAO) Activators? A Theoretical Case Study involving rac-Me2Si(η5-C9H6)2Zr (SBIZr) Complexes.

Activation of SBIZrMe2 or SBIZrMeCl and a sheet model for an active component of hydrolytic MAO, (MeAlO)16(Me3Al)6, (16,6) has been studied by DFT. Contact ion-pair formation occurs through the intermediacy of SBIZrMe(Cl) or SBIZrMe2 reacting with sheet 16,6 to furnish SBIZrMe-µ-X(MeAlO)16(Me3Al)6 (2, X=Me, Cl). Contact ion-pairs 2 would be in equilibrium with heterodinuclear catalyst precursors [SBIZrMe2AlMe2][(MeAlO)16(Me3Al)6X] (3 (X=Me, Cl) through reversible binding of Me3Al at higher Al : Zr ratios. Calculations show that formation of ion-pairs 3 from contact ion-pairs 2 is more favourable for the SBIZr compared with the parent Cp2Zr complexes. TD-DFT calculations were conducted on relevant SBIZr complexes to relate the results to earlier spectroscopic studies of catalyst activation using UV-Vis spectroscopy. Finally, propene insertion into ion-pairs 2, SBIZrMe-µ-MeB(C6F5)3 (6) and [SBIZrMe][B(C6F5)4] (7) was studied at M06-2X/TZVP level of theory. These studies suggest that contact ion-pairs 2 are significantly less reactive towards insertion than 6 or 7, in disagreement with experiment.

In Vivo Glucose Transporter-2 Regulation of Dorsomedial Versus Ventrolateral VMN Astrocyte Metabolic Sensor and Glycogen Metabolic Enzyme Gene Expression in Female Rat.

Astrocyte glycogenolysis shapes ventromedial hypothalamic nucleus (VMN) regulation of glucostasis in vivo. Glucose transporter-2 (GLUT2), a plasma membrane glucose sensor, controls hypothalamic primary astrocyte culture glycogen metabolism in vitro. In vivo gene silencing tools and single-cell laser-catapult-microdissection/multiplex qPCR techniques were used here to examine whether GLUT2 governs dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN astrocyte metabolic sensor and glycogen metabolic enzyme gene profiles. GLUT2 gene knockdown diminished astrocyte GLUT2 mRNA in both VMN divisions. Hypoglycemia caused GLUT2 siRNA-reversible up-regulation of this gene profile in the VMNdm, but down-regulated VMNvl astrocyte GLUT2 transcription. GLUT2 augmented baseline VMNdm and VMNvl astrocyte glucokinase (GCK) gene expression, but increased (VMNdm) or reduced (VMNvl) GCK transcription during hypoglycemia. GLUT2 imposed opposite control, namely stimulation versus inhibition of VMNdm or VMNvl astrocyte 5'-AMP-activated protein kinase-alpha 1 and -alpha 2 gene expression, respectively. GLUT2 stimulated astrocyte glycogen synthase (GS) gene expression in each VMN division. GLUT2 inhibited transcription of the AMP-sensitive glycogen phosphorylase (GP) isoform GP-brain type (GPbb) in each site, yet diminished (VMNdm) or augmented (VMNvl) astrocyte GP-muscle type (GPmm) mRNA. GLUT2 enhanced VMNdm and VMNvl glycogen accumulation during euglycemia, and curbed hypoglycemia-associated VMNdm glycogen depletion. Results show that VMN astrocytes exhibit opposite, division-specific GLUT2 transcriptional responsiveness to hypoglycemia. Data document divergent GLUT2 control of GCK, AMPK catalytic subunit, and GPmm gene profiles in VMNdm versus VMNvl astrocytes. Ongoing studies seek to determine how differential GLUT2 regulation of glucose and energy sensor function and glycogenolysis in each VMN location may affect local neuron responses to hypoglycemia.

An interactive R-based custom quantification program for semi-quantitative analysis of triacylglycerols in bovine milk.

The R programming language, RStudio, and open-source software solutions for analysis of liquid chromatography-mass spectrometry (LC-MS) data have been used with user-written R-based custom quantification programs (CQP) for semi-quantification of triacylglycerols (TAGs) in bovine milk lipid extracts. Using the peak-finding capabilities of the package "xcms" in RStudio, peaks were integrated, and retention times aligned, normalized, and then used for semi-quantitative analysis of a custom set of four extraction internal standards (EISs) and 29 TAG regioisomers using the choice of four analytical internal standards (AISs). Alternating stereospecific numbering (sn) 1,3 TAG regioisomers (standards 1, 3, and 5 of six calibration standards) and sn-1,2 TAG regioisomers (standards 2, 4, and 6 of six standards) were used to make a set of six calibration standards, which were used for quantification using a linear fit model, polynomial fit model, power fit model, level-bracketed linear fit, replicate-bracketed polynomial fit, replicate-bracketed power fit, and replicate- and level-bracketed linear fit and response factors. For example, the linear fit for EIS1 gave an unacceptable coefficient of determination (CoD), r2 = 0.9616, whereas the polynomial fit gave r2 = 0.9908 and the power fit gave r2 = 0.9928, while the double-bracketed linear fit gave CoDs of r2 = 0.9960, 0.9848, and 0.9781 for the three brackets, yet gave the least % difference to known calibration concentrations. For unparalleled transparency, the CQP produced webpages that allowed every step in the data processing and quantification sequence to be verified and reproduced, and contained interactive figures. The data are publicly available using a digital object identifier (DOI). The R code can be downloaded and used with the downloadable data to reproduce the results, to modify the code and further customize the results, or to copy and paste and adapt the code to other quantification applications.

Arsenobetaine amide: a novel arsenic species detected in several mushroom species.

The total arsenic mass fraction as well as the arsenic speciation were studied in four different mushroom species with inductively coupled plasma mass spectrometry and high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry, respectively. Arsenic mass fractions detected in the mushrooms were covering a range from 0.3 to 22 mg As kg-1 dry mass. For the arsenic speciation, species like arsenobetaine, inorganic arsenic, or dimethylarsinic acid were found, which are commonly detected in mushrooms, but it was also proven that the recently discovered novel compound homoarsenocholine is present in Amanita muscaria and Ramaria sanguinea. Moreover, a previously unidentified arsenic species was isolated from Ramaria sanguinea and identified as trimethylarsonioacetamide, or in short: arsenobetaine amide. This new arsenical was synthesized and verified by spiking experiments to be present in all investigated mushroom samples. Arsenobetaine amide could be an important intermediate to further elucidate the biotransformation pathways of arsenic in the environment.

Micro simulated moving bed chromatography-mass spectrometry as a continuous on-line process analytical tool.

Continuous manufacturing is becoming increasingly important in the (bio-)pharmaceutical industry, as more product can be produced in less time and at lower costs. In this context, there is a need for powerful continuous analytical tools. Many established off-line analytical methods, such as mass spectrometry (MS), are hardly considered for process analytical technology (PAT) applications in biopharmaceutical processes, as they are limited to at-line analysis due to the required sample preparation and the associated complexity, although they would provide a suitable technique for the assessment of a wide range of quality attributes. In this study, we investigated the applicability of a recently developed micro simulated moving bed chromatography system (µSMB) for continuous on-line sample preparation for MS. As a test case, we demonstrate the continuous on-line MS measurement of a protein solution (myoglobin) containing Tris buffer, which interferes with ESI-MS measurements, by continuously exchanging this buffer with a volatile ammonium acetate buffer suitable for MS measurements. The integration of the µSMB significantly increases MS sensitivity by removing over 98% of the buffer substances. Thus, this study demonstrates the feasibility of on-line µSMB-MS, providing a versatile PAT tool by combining the detection power of MS for various product attributes with all the advantages of continuous on-line analytics.

Study of metalation of thioredoxin by gold(I) therapeutic compounds using combined liquid chromatography/capillary electrophoresis with inductively coupled plasma/electrospray MS/MS detection.

The reactivity of thioredoxin (Trx1) with the Au(I) drug auranofin (AF) and two therapeutic N-heterocyclic carbene (NHC)2-Au(I) complexes (bis [1-methyl-3-acridineimidazolin-2-ylidene]gold(I) tetrafluoroborate (Au3BC) and [1,3-diethyl-4,5-bis(4methoxyphenyl)imidazol-2-ylidene]gold(I) (Au4BC)) was investigated. Direct infusion (DI) electrospray ionization (ESI) mass spectrometry (MS) allowed information on the structure, stoichiometry, and kinetics of formation of Trx-Au adducts. The fragmentation of the formed adducts in the gas phase gave insights into the exact Au binding site within the protein, demonstrating the preference for Trx1 Cys32 or Cys35 of AF or the (NHC)2-Au(I) complex Au3BC, respectively. Reversed-phase HPLC suffered from the difficulty of elution of gold compounds, did not preserve the formed metal-protein adducts, and favored the loss of ligands (phosphine or NHC) from Au(I). These limitations were eliminated by capillary electrophoresis (CE) which enabled the separation of the gold compounds, Trx1, and the formed adducts. The ICP-MS/MS detection allowed the simultaneous quantitative monitoring of the gold and sulfur isotopes and the determination of the metallation extent of the protein. The hyphenation of the mentioned techniques was used for the analysis of Trx1-Au adducts for the first time.

Derivatization procedure of estradiol with a combination of MPDNP-F and 4-dimethylaminopyridine to generate product ion containing estradiol-skeleton for reliable determination of its serum/plasma concentrations by LC/ESI-MS/MS.

The quantification of serum/plasma estradiol (E2) is useful for the diagnosis, pathological analysis, and monitoring of the therapeutic efficacy of estrogen-dependent diseases. In this study, an improved derivatization method using 1-(2,4-dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium iodide (MPDNP-F) was developed and combined with liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the sensitive and specific quantification of the serum/plasma E2. In the new method, the reaction time was reduced to 15 min from 90 min (two-step reaction in the previous method) by the direct reaction of MPDNP-F with E2 at 60°C in the presence of 4-dimethylaminopyridine (DMAP). DMAP served as the organic catalyst and had a less negative effect on the LC/ESI-MS/MS instrument compared to the non-volatile inorganic salt (NaHCO3), which was used in the previous method. The collision-induced dissociation of the molecular cation ([M]+) of the resulting derivative provided a product ion containing the E2-skeleton ([M-NO2-H]+), which significantly enhanced the assay sensitivity and specificity; compared to the dansyl chloride derivatization, which is the currently most-used derivatization procedure for the LC/ESI-MS/MS assays of E2, the MPDNP-F derivatization had significantly fewer interfering peaks and a clear and flat baseline in the serum sample analysis. The MPDNP-F derivatization-LC/ESI-MS/MS method enabled the precise and accurate quantification of E2 even at a 5.0 pg/mL concentration (lower limit of quantification) with a small sample volume (100 µL of serum/plasma) and had a tolerance for the matrix effect. This method was also proven to serve as a more sensitive and specific alternative to the clinically used chemiluminescence enzyme immunoassay.

Development of an analytical method to quantify N-acyl-homoserine lactones in bacterial cultures, river water, and treated wastewater.

N-Acyl-homoserine lactones (AHL) play a major role in the communication of Gram-negative bacteria. They influence processes such as biofilm formation, swarming motility, and bioluminescence in the aquatic environment. A comprehensive analytical method was developed to elucidate the "chemical communication" in pure bacterial cultures as well as in the aquatic environment and engineered environments with biofilms. Due to the high diversity of AHLs and their low concentrations in water, a sensitive and selective LC-ESI-MS/MS method combined with solid-phase extraction was developed for 34 AHLs, optimized and validated to quantify AHLs in bacterial conditioned medium, river water, and treated wastewater. Furthermore, the developed method was optimized in terms of enrichment volume, internal standards, limits of detection, and limits of quantification in several matrices. An unanticipated variety of AHLs was detected in the culture media of Pseudomonas aeruginosa (in total 8 AHLs), Phaeobacter gallaeciensis (in total 6 AHLs), and Methylobacterium mesophilicum (in total 15 AHLs), which to our knowledge have not been described for these bacterial cultures so far. Furthermore, AHLs were detected in river water (in total 5 AHLs) and treated wastewater (in total 3 AHLs). Several detected AHLs were quantified (in total 24) using a standard addition method up to 7.3±1.0 µg/L 3-Oxo-C12-AHL (culture media of P. aeruginosa).

Microbiome of seventh-century old Parsurameswara stone monument of India and role of desiccation-tolerant cyanobacterium Lyngbya corticicola on its biodeterioration.

The Parsurameswara stone monument, built in the seventh century, is one of the oldest stone monuments in Odisha, India. Metagenomic analysis of the biological crust samples collected from the stone monument revealed 17 phyla in the microbiome, with Proteobacteria being the most dominant phylum, followed by cyanobacteria. Eight cyanobacteria were isolated. Lyngbya corticicola was the dominant cyanobacterium in all crust samples and could tolerate six months of desiccation in vitro. With six months of desiccation, chlorophyll-a decreased; however, carotenoid and cellular carbohydrate contents of this organism increased in the desiccated state. Resistance to desiccation, high carotenoid content, and effective trehalose biosynthesis in this cyanobacterium provide a distinct advantage over other microbiomes. Comparative metabolic profiles of the biological crust and L. corticicola show strongly corrosive organic acids such as dichloroacetic acid, which might be responsible for the biocorrosion of stone monuments.

Improvement of Alginate Extraction from Brown Seaweed (Laminaria digitata L.) and Valorization of Its Remaining Ethanolic Fraction.

This study aimed to improve the conventional procedure of alginate isolation from the brown seaweed (Laminaria digitata L.) biomass and investigate the possibility of further valorization of the ethanolic fraction representing the byproduct after the degreasing and depigmentation of biomass. The acid treatment of biomass supported by ultrasound was modeled and optimized regarding the alginate yield using a response surface methodology based on the Box-Behnken design. A treatment time of 30 min, a liquid-to-solid ratio of 30 mL/g, and a treatment temperature of 47 °C were proposed as optimal conditions under which the alginate yield related to the mass of dry biomass was 30.9%. The use of ultrasonic radiation significantly reduced the time required for the acid treatment of biomass by about 4 to 24 times compared to other available conventional procedures. The isolated alginate had an M/G ratio of 1.08, which indicates a greater presence of M-blocks in its structure and the possibility of forming a soft and elastic hydrogel with its use. The chemical composition of the ethanolic fraction including total antioxidant content (293 mg gallic acid equivalent/g dry weight), total flavonoid content (14.9 mg rutin equivalent/g dry weight), contents of macroelements (the highest content of sodium, 106.59 mg/g dry weight), and microelement content (the highest content of boron, 198.84 mg/g dry weight) was determined, and the identification of bioactive compounds was carried out. The results of ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis confirmed the presence of 48 compounds, of which 41 compounds were identified as sugar alcohol, phenolic compounds, and lipids. According to the 2,2-diphenyl-1-picrylhydrazyl assay, the radical scavenging activity of the ethanolic fraction (the half-maximal inhibitory concentration of 42.84 ± 0.81 µg/mL) indicated its strong activity, which was almost the same as in the case of the positive control, synthetic antioxidant butylhydroxytoluene (the half-maximal inhibitory concentration of 36.61 ± 0.79 µg/mL). Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus) were more sensitive to the ethanolic fraction compared to Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei). The obtained results indicated the possibility of the further use of the ethanolic fraction as a fertilizer for plant growth in different species and antifouling agents, applicable in aquaculture.

Determination of three C18-oxygenated steroids in adrenal lesion segments in primary aldosteronism by super-selective adrenal venous sampling and LC/ESI-MS/MS.

Super-selective adrenal venous sampling (ssAVS) can collect the adrenal tributary venous blood in the aldosterone (ALD)-hypersecreting segments in primary aldosteronism. The concentrations of the C18-oxygenated steroids, especially 18-oxocortisol (18-oxoF), in the lesion segments might be more useful indices than those in the peripheral or adrenal central veins (current candidate indexes) for the differential diagnosis of unilateral ALD-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). To verify this hypothesis, we developed a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for simultaneously quantifying ALD, 18-oxoF and 18-hydroxycortisol in the adrenal tributary venous serum sample collected by ssAVS (ssAVS serum) and compared their concentrations between APA and BAH patients. Only deproteinization was required for a 10 µl sample prior to the LC/ESI-MS/MS analysis. Endogenous corticoids did not interfere with the quantifications, and the intra-assay and interassay precisions (≤ 8.3%) and accuracies (94.2-102.7%) were acceptable. The clinical study revealed that the 18-oxoF concentration was significantly higher in the ALD-producing tumor tissues (from APA patients) than in the hyperplastic tissues (from BAH patients). However, in conclusion, the 18-oxoF concentration in the ssAVS serum sample can be a rough indication but cannot be decisive for the differential diagnosis between APA and BAH owing to the significant individual difference.

Phenolic Compound Profiles, Antioxidant and Cytoprotective Activity of Elaeagnus conferta Roxb. Fruit.

In this paper, three varieties of Elaeagnus conferta Roxb fruits prepared by ultrasonic-assisted extraction from a subtropical region southwest of China were utilized as raw materials to investigate their phenolic profiles, antioxidant activities, and protective effects on injured human umbilical vein endothelial cells (HUVECs). The ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) findings revealed that fifteen substances, including seven phenolic acids, seven flavonoids, and one gallic acid derivative, were discovered. The dihydromyricetin, ellagic acid, gallic acid were the predominant phenolic compounds in all E.conferta fruits. These E.conferta fruits extracts shown excellent antioxidant activity varied from 2.258 ± 0.03 ~ 7.844 ± 0.39 µM Trolox/g and protective effect on HUVECs injured by H2O2 through decrease the level of ROS, MDA, LDH and enhance the SOD level. These finding indicate that E.conferta is a valuable source of high-capacity antioxidants that might be used as an alternative material for food industries.

Variation in Phenolic Content, Antioxidant Activity and Alpha-amylase and Acetylcholinesterase Inhibitory Capacities of Different Extracts from Tunisian Satureja barceloi (Willk) L.

The antioxidant activity and the anti-α-amylase and anti-acetylcholinesterase capacities of secondary metabolites from different organs (roots, stems, leaves and flowers) of Tunisian Satureja barceloi were determined. The variation in the distribution of phenolic metabolites among roots, stems, leaves and flowers extracts of S. barceloi with various solvent systems (methanol, ethyl acetate, hexane and distilled water) has not been characterized before. Significant variation of phenolic compounds was observed according to organs rather than to extracting solvents. The analyzed organs show a high level of phenolic compounds although the stems contains the highest total polyphenols (132.53±0.48 mg AGE/g Ex), flavonoids (48.99±0.65 mg RE/g Ex) and flavonols (34.93±0.29 mg QE/g Ex) contents. The phenolic fraction was dominated by sagerinic acid, caffeic acid glucoside and epigallocatechin, detected using HPLC-PDA/ESI-MS. The antioxidant activity of all extracts, evaluated by four in vitro tests, was high and varied significantly according to the type of solvent used and the plant organ. The aqueous extracts of leaves exhibited the greatest inhibitory effect on alpha-amylase while the methanolic extract of leaves and stems revealed the most important acetylcholinesterase inhibitory effect. Hence, S. barceloi extracts could be used as a source of various bioactive molecules in pharmaceutical industry.

Anti-Culex pipiens activity of different pomegranate cultivars and determination of their bioactive compounds using LC-MS profiling.

INTRODUCTION: Pomegranate (Punica granatum L.) peels are rich in various bioactive compounds. Characterization of these compounds is crucial for the utilization of peel waste in industrial processing. OBJECTIVE: The study aimed (1) to establish and compare the metabolic profiles of the peel of seven pomegranate cultivars and (2) to identify bioactive compounds contributing to the larvicidal activity against the third instar larvae of Culex pipiens. MATERIALS AND METHODS: UPLC-ESI-MS/MS was utilized to analyze peel methanol extracts of different pomegranate cultivars. The larvicidal activity was determined by calculating the larval mortality among the third instar larvae of C. pipiens. Multivariate data analysis was conducted to identify the metabolites that exhibited a larvicidal effect. RESULTS: A total of 24 metabolites, including hydrolyzable tannins, flavonoids, and alkaloids, were tentatively identified in both negative and positive ionization modes. The extract of cultivar 'Black' exhibited the most potent larvicidal effect with LC50 values of 185.15, 156.84, and 138.12 ppm/mL after 24, 48, and 72 h of treatment, respectively. By applying chemometric techniques, the larvicidal activity could be directly correlated to the bioactive compounds punicalagin, quercetin-O-rhamnoside, quercetin-O-pentoside, and galloyl-HHDP-glucose. CONCLUSION: The present study implemented UPLC-ESI-MS/MS and chemometric techniques as potential tools for metabolomics analysis and differentiation between peels of different pomegranate cultivars. In addition, cultivar 'Black' extract could be a promising natural insecticide against mosquitoes since it is rich in bioactive compounds with larvicidal activity.