Metabolic profiles and non-targeted LC-MS/MS approach as a complementary tool to targeted analysis in assessment of plant exposure to pesticides.

Liquid chromatography coupled with tandem mass spectrometry was employed for the detection of pesticides (thiamethoxam, lambda-cyhalothrin, deltamethrin, and metalaxyl) and their metabolites in Raphanus sativus var. longipinnatus exposed to these compounds under experimental conditions. Metalaxyl (0.008 mg/kg), metalaxyl acid (0.009 mg/kg), and (+)-trans-chrysanthemic acid (0.098 mg/kg) were identified in the plants exposed to the individual pesticides and their metabolites. Non-targeted analysis revealed the presence of thiamethoxam, lambda-cyhalothrin, and deltamethrin metabolites in plants exposed to these substances, despite the fact that the pesticide concentrations were below the analytical method's limit of quantification (0.005-0.006 mg/kg). Based on the non-targeted screening, non-specific (leucine and tyramine) and specific (epinephrine, dopamine, tryptamine, and serotonin) markers of plant exposure to the mentioned stress-inducing compounds were detected. These findings prove that non-targeted analysis is an indispensable tool for determining plants' exposure to pesticides, even when the parent compound has been completely metabolized.

Facile preparation of ethanediamine-ß-cyclodextrin modified capillary column for electrochromatographic enantioseparation of Dansyl amino acids.

Herein, the fabrication of a fascinating multifunctional cyclodextrin (CD) chiral stationary phase and its chiral separation performance in capillary electrochromatography are proposed. A facile interfacial polymerization was used to anchor ethanediamine-ß-cyclodextrin (EDA-ß-CD) polymerized with trimesoyl chloride (TMC) and to form the chiral stationary phase (CSP) composite onto the surface wall of the capillary. The characters of prepared columns were confirmed by Fourier transform infrared spectroscopy (FT-IR), X-ray Photoelectron Spectrometer (XPS), scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). This novel CSP offers multi-typical interactions including hydrogen bonding, π-interaction, hydrophobic and electrostatic interaction as well as steric effects which contribute to prominent chiral recognition for Dansyl-DL-amino acids in CEC modes. The EDA-ß-CD modified column showed eminent enantioseparation performance towards five Dansyl-DL-amino acids (the DL-forms of valine, threonine, leucine, phenylalanine, serine). Besides, the prepared columns were perfectly reproducible and stable. The relative standard deviations of the enantiomer retention times for intra-day (n = 5), inter-day (n = 3) runs and column-to-columns (n = 3) are below 0.54%, 1.35% and 4.89%, individually. This innovative chiral stationary phase shows a broader application view and scope in chiral recognition domain.

Physicochemical and Pharmacokinetic Evaluation of Spray-Dried Coformulation of Salvia miltiorrhiza Polyphenolic Acid and L-Leucine with Improved Bioavailability.

Background:Salvia miltiorrhiza polyphenolic acid (SMPA) is effective in the treatment of cardiovascular diseases and currently it is administered orally or intravenously. However, SMPA is poorly absorbed orally and quickly eliminated in vivo. A long-term frequent intravenous administration leads to poor patient compliance. Therefore, it is urgently demanded to find a new alternative route of noninjection drug delivery system for SMPA. Methods: Two dry powder inhalation (DPI) formulations of spray-dried SMPA formulation (P1) and spray-dried SMPA-L-leucine formulation (P2) were prepared by spray drying method and their physicochemical properties were assessed by thermogravimetric analysis, X-ray diffraction, scanning electron microscopy, particle size distribution analysis, and in vitro aerodynamic analysis. Moreover, In vitro cytotoxicity of SMPA and P2 was conducted with NR8383 cells. In vivo pharmacokinetics were carried out by Penn-Century endotracheal intubation technique to deliver P2 to the lungs of rats. Results and Conclusions: The moisture content of P1 and P2 were 5.81% ± 0.005%, and 4.08% ± 0.002%, respectively. P1 and P2 were in an amorphous state. Moreover, P1 had slightly corrugated surfaces, whereas P2 exhibited severely corrugated surfaces with invagination due to the presence of L-leucine. In addition, there were more hollow particles with smooth surface in P1 than that in P2. Compared with P1, P2 has shown optimal physical particle size and aerosolization behavior with D (v, 50) of 2.64 ± 0.01 µm and fine particle fraction of 37.55% ± 2.63%. The findings of in vitro cytotoxicity showed that P2 did not inhibit cell viability and could be safe for pulmonary administration. The absolute bioavailability of salvianolic acid B (Sal B) for pulmonary administration was 19.15% ± 7.44%, which is significantly higher than the oral bioavailability of Sal B (<5.56%). In this study, we have shown the feasibleness of pulmonary administration of SMPA in the form of DPIs for systemic delivery to treat cardiovascular diseases.

A peculiar chromatographic selectivity of porous graphitic carbon during the separation of dileucine isomers.

Porous graphitic carbon is a versatile stationary phase for high-performance liquid chromatography which performs especially well for isomeric separations. Shape-sensitivity of the stationary phase is caused by a steric effect when a molecule interacts with a flat carbon surface. It follows that branched, non-flat molecules are eluted much earlier than flat or linear molecules. In this short communication we show that if a molecule has a highly ionizable group, the "shape" of a molecule part which is farther from the ionizable group affects retention much more than the "shape" of a molecule part which is closer to the ionizable group. Dipeptides which consist of tert-leucine and norleucine were used as an example. Basic and acidic eluents were used. Retention strongly depends on whether a norleucine or tert-leucine residual is located near the non-ionized side in an eluent for both basic and acidic eluents. A residual located on the opposite side is less important. To investigate the possible causes of this peculiar retention behavior we compared the retention behavior of these dipeptides for porous graphitic carbon with the behavior for other types of stationary phases and with the calculated physicochemical properties. Strong and complex dependence of elution order on a mobile phase composition is demonstrated. The separation of other dileucine isomers is also considered. The applicability of porous graphitic carbon for the separation of complex mixtures of isomeric peptides is discussed.

Zwitterionic codeine-derived methacrylate monoliths for enantioselective capillary electrochromatography of chiral acids and chiral bases.

Thiol-ene click reaction of N-acetyl-L-cysteine methyl ester to codeine, followed by reaction with allyl isocyanate and hydrolysis to the corresponding zwitterionic chiral selector and its subsequent bonding to the surface of a methacrylate monolith provided a new chiral capillary column for enantiomer separation of chiral acids and chiral bases. First, the epoxy groups of a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were converted into amine residues, followed by reaction with allylglycidyl ether. In this way, a spacer arm was bonded to the surface before coating and cross-linking poly(3-mercaptopropyl methylsiloxane) (PMPMS) via radical addition (thiol-ene click reaction) to the surface. In order to improve the performance of the monolithic chiral stationary phase, thio ether and residual thiol groups were oxidized to sulfonyl and sulphonate groups, respectively. This novel chiral stationary phase (CSP) was evaluated by capillary electrochromatography (CEC) using two chiral model compounds, namely N-3,5-dinitrobenzoyl-R,S-leucine (retained by anion-exchange mechanism) and mefloquine (by cation-exchange process). The ion-exchange retention mechanism on the CSP was characterized for these two counterionic model solutes by varying the mobile phase composition, including the nature of solvents, the concentration of counter-ions and co-ions, and the acid-to-base ratio. A series of chiral ß-blockers and amino acid derivatives was used to further check the performance of the modified monolith under the optimal conditions. Several enantiomers were baseline resolved with reasonable peak efficiencies (up to 60,000 theoretical plates per meter for the second eluted enantiomer).

Hydrophobic Amino Acid Content in Onions as Potential Fingerprints of Geographical Origin: The Case of Rossa da Inverno sel. Rojo Duro.

In this study, we were interested in comparing the amino acid profile in a specific variety of onion, Rossa da inverno sel. Rojo Duro, produced in two different Italian sites: the Cannara (Umbria region) and Imola (Emilia Romagna region) sites. Onions were cultivated in a comparable manner, mostly in terms of the mineral fertilization, seeding, and harvesting stages, as well as good weed control. Furthermore, in both regions, the plants were irrigated by the water sprinkler method and subjected to similar temperature and weather conditions. A further group of Cannara onions that were grown by micro-irrigation was also evaluated. After the extraction of the free amino acid mixture, an ion-pairing reversed-phase (IP-RP) HPLC method allowed for the separation and the evaporative light scattering detection of almost all the standard proteinogenic amino acids. However, only the peaks corresponding to leucine (Leu), phenylalanine (Phe), and tryptophan (Trp), were present in all the investigated samples and they were unaffected from the matrix interfering peaks. The use of the beeswarm/box plots revealed that the content of Leu and Phe were markedly influenced by the geographical origin of the onions (with *** p.

Depsidomycins B and C: New Cyclic Peptides from a Ginseng Farm Soil-Derived Actinomycete.

LC/MS-based chemical profiling of a ginseng farm soil-derived actinomycete strain, Streptomyces sp. BYK1371, enabled the discovery of two new cyclic heptapeptides, depsidomycins B and C (1 and 2), each containing two piperazic acid units and a formyl group at their N-terminus. The structures of 1 and 2 were elucidated by a combination of spectroscopic and chemical analyses. These new compounds were determined to possess d-leucine, d-threonine, d-valine, and S-piperazic acid based on the advanced Marfey's method and a GITC (2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate) derivatization of their hydrolysates, followed by LC/MS analysis. Depsidomycins B and C displayed significant antimetastatic activities against metastatic breast cancer cells (MDA-MB-231).

In-situ functionalized monolithic polysiloxane-polymethacrylate composite materials from polythiol-ene double click reaction in capillary column format for enantioselective nano-high-performance liquid chromatography.

This work reports on the proof-of-principle of preparation of novel one step in-situ functionalized monolithic polysiloxane-polymethacrylate composite materials in capillary columns for enantioselective nano-HPLC using a thiol-ene click reaction. Quinine carbamate as functional monomer and ethylene dimethacrylate as crosslinker were both used as ene components in a thermally initiated double click-type polymerization reaction with poly(3-mercaptopropyl)methylsiloxane as thiol component in presence of 1-propanol as porogenic solvent. Elemental analysis and on-capillary fluorescence measurement proved the successful incorporation of the functional chiral monomer into the polymer. Scanning electron microscopy images revealed a macroporous polymer morphology which is typical for a nucleation and growth mechanism of pore formation. The individual microglobules appear relatively spherical and smooth indicating a non-porous nature. Nano-HPLC experiments of the chiral monolithic capillary column provided successful enantiomer separation of N-3,5-dinitrobenzoylleucine as test compound in polar organic elution mode clearly documenting the successful implementation of the proposed concept towards new functionalized monolithic composite materials.

Three new amino acid derivatives from edible mushroom Pleurotus ostreatus.

Three new amino acid derivatives, oxalamido-L-phenylalanine methyl ester (1), oxalamido-L-leucine methyl ester (2), and lumichrome hydrolyzate (3), together with nine known compounds (4-12), were isolated from the solid culture of edible mushroom Pleurotus ostreatus. Their structures were elucidated on the basis of extensive spectroscopic analysis. The absolute configurations of 1 and 2 were established by the chiral synthesis and confirmed by circular dichroism (CD) analysis of their total synthesis products and natural isolates. All new compounds were evaluated for their antioxidant effects, antimicrobial activities, and cytotoxic activity. Compounds 1-3 showed weak antifungal activities against Candida albicans with minimum inhibitory concentration (MIC) value of 500 µg/ml.

Chiral HPLC Separation and Modeling of Four Stereomers of DL-Leucine-DL-Tryptophan Dipeptide on Amylose Chiral Column.

Chiral high-performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL-leucine-tryptophan DL-dipeptide on AmyCoat-RP column are described. The mobile phase applied was ammonium acetate (10 mM)-methanol-acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL-, DD-, DL-, and LD- stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0-2.3 and 5.6-14.0 µg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π-π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL-leucine-DL-tryptophan in unknown matrices. Chirality 28:642-648, 2016. © 2016 Wiley Periodicals, Inc.

Rhizoleucinoside, a Rhamnolipid-Amino Alcohol Hybrid from the Rhizobial Symbiont Bradyrhizobium sp. BTAi1.

Rhizoleucinoside (1), a unique rhamnolipid-amino alcohol hybrid, was isolated from the rhizobial symbiont bacterium Bradyrhizobium sp. BTAi1. Compound 1 features a rare rhamnolipid core attached to an unprecedented leucinol moiety. Its structure and absolute configuration were determined by spectroscopic analysis, tandem mass spectrometry, chemical degradation, and application of the Marfey's method. Compound 1 possesses moderate cytotoxicity to BV-2 murine microglia and highly aggressive proliferating immortalized (HAPI) rat microglia cells.

The Natural Product N-Palmitoyl-l-leucine Selectively Inhibits Late Assembly of Human Spliceosomes.

The spliceosome is a dynamic complex of five structural RNAs and dozens of proteins, which assemble together to remove introns from nascent eukaryotic gene transcripts in a process called splicing. Small molecules that target different components of the spliceosome represent valuable research tools to investigate this complicated macromolecular machine. However, the current collection of spliceosome inhibitors is very limited. To expand the toolkit we used a high-throughput in vitro splicing assay to screen a collection of pre-fractions of natural compounds derived from marine bacteria for splicing inhibition. Further fractionation of initial hits generated individual peaks of splicing inhibitors that interfere with different stages of spliceosome assembly. With additional characterization of individual peaks, we identified N-palmitoyl-l-leucine as a new splicing inhibitor that blocks a late stage of spliceosome assembly. Structure-activity relationship analysis of the compound revealed that length of carbon chain is important for activity in splicing, as well as for effects on the cytological profile of cells in culture. Together these results demonstrate that our combination of in vitro splicing analysis with complex natural product libraries is a powerful strategy for identifying new small molecule tools with which to probe different aspects of spliceosome assembly and function.

Metabolites of Microcystis aeruginosa bloom material from Lake Kinneret, Israel.

Six new metabolites, micropeptin KT1042, microguanidine KT636, aeruginosins KT608A, KT608B, and KT650, and pseudoaeruginosin KT554, were isolated along with the known micropeptins SF909 and HM978, cyanopeptolin S, anabaenopeptin F, and the two isomers of planktocyclin-S-oxide from a bloom material collected from Lake Kinneret, Israel, in March 2005. The structure elucidation and biological activity of the six new natural products isolated from this bloom material and the related aeruginosin GH553 are described.

Multi-wavelength light emitting diode array as an excitation source for light emitting diode-induced fluorescence detection in capillary electrophoresis.

A multi-wavelength LED array was used as an excitation source for in-column fiber-optic LED-induced fluorescence for CE. The light source consisted of a multi-wavelength LED array consisting of three different LEDs (430, 450 and 480 nm), a focusing lens and a gradient index lens group. The LED beam was collimated and reshaped with the gradient index lens group for coupling the LED light source into a single-mode optical fiber. In addition, the luminance and stability of the LED light source was improved by powering the LED under constant current at enhanced voltages. The benefits of this system were demonstrated by the simultaneous determination of FITC-labeled L-asparagine (Ex/Em 488/520 nm), 4-fluoro-7-nitro-2,1,3-benzoxadiazole-labeled epinephrine (Ex/Em 468/530 nm) and 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde-labeled L-leucine (Ex/Em 440/530 nm). Detection limits of L-asparagine, epinephrine and L-leucine were estimated to be 0.8 x 10(-9), 12.0 x 10(-8) and 4.0 x 10(-8) M (S/N=3), respectively. The RSDs (n=6) for migration time and peak area were better than 0.71 and 0.92%, respectively. The performance of the developed multi-wavelength LED excitation source was compared to the use of a single-wavelength LED and found to provide superior sensitivity for the three fluorophores used in this study.

Synthesis and chromatographic properties of a chiral stationary phase derived from bovine serum albumin immobilized on magnesia-zirconia using phosphonate spacers.

A novel bovine serum albumin (BSA)-modified magnesia-zirconia stationary phase was prepared using the sodium salt of cis-(3-methyloxiranyl)phosphonic acid (fosfomycin) as spacer and glutaraldehyde as coupler. Baseline separation of six derivatized amino acids (DNB-Leu, Dansyl-Val, etc.) was achieved on this column using ammonium acetate buffer-isopropanol mobile phase at a flow rate of 1.0 mL/min. The effects of mobile phase composition, eluent pH value, column temperature, and flow rate on the retention and separation of chiral compounds were also investigated. The BSA chiral stationary phase (BSA-CSP) was relatively stable under experimental conditions. The coupling reaction in this method was mild, reliable, and reproducible; thus it was also suitable for the immobilization of various biopolymers with amino groups in the preparation of chromatography stationary phases.

Urukthapelstatin A, a novel cytotoxic substance from marine-derived Mechercharimyces asporophorigenens YM11-542. I. Fermentation, isolation and biological activities.

Urukthapelstatin A, a novel cyclic peptide, was isolated from the cultured mycelia of marine-derived Thermoactinomycetaceae bacterium Mechercharimyces asporophorigenens YM11-542. The peptide was purified by solvent extraction, silica gel chromatography, ODS flash chromatography, and finally by preparative HPLC. Urukthapelstatin A dose-dependently inhibited the growth of human lung cancer A549 cells with an IC(50) value of 12 nM. Urukthapelstatin A also showed potent cytotoxic activity against a human cancer cell line panel.

Characterization of a chiral stationary phase by HR/MAS NMR spectroscopy and investigation of enantioselective interaction with chiral ligates by transferred NOE.

The surface chemistry of a chiral stationary phase (CSP) with a (tert-butyl carbamoyl) quinine selector immobilized on thiol-modified silica has been characterized by (1)H HR/MAS NMR and (29)Si CP/MAS NMR spectroscopy. The mostly well-resolved (1)H signals could be assigned to stem from the surface-bound selector and the latter suggested a bi- and trifunctional silane linkage. Suspended-state NMR spectroscopy thus proved a well-characterized surface chemistry as proposed. To study chiral recognition phenomena in the presence of the CSP, (1)H HR/MAS 2D transfer NOESY investigations in methanol-d(4) have been undertaken with various solutes including N-3,5-dinitrobenzoyl derivatives of leucine (DNB-Leu) and N-acetyl phenylalanine (Ac-Phe). Both (R)- and (S)-enantiomers of DNB-Leu and Ac-Phe interacted with the tBuCQN-CSP as indicated by negative cross-peaks in the trNOESY spectra, while the 2D NOESY of the dissolved solutes in absence of the chiral stationary phase showed positive cross-peaks. The intensities of the trNOE cross-peaks were much stronger for the (S)-enantiomers. This stereoselectivity paralleled the experimental chromatographic behavior, where the (S)-enantiomers revealed stronger binding and retention on the tBuCQN-CSP as well. Hence, we were able to correlate the retention behavior to the trNOE NMR spectroscopic data in a qualitative manner.

Combination of cyclodextrins and polymeric surfactants for chiral separations.

A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) method was applied to the enantioseparation of three binaphthyl derivatives using neutral CDs (i.e., beta- and gamma-CD) in combination with various chiral amino acid-based polymeric surfactants (PSs). Both the D- and L-configurations of poly(sodium N-undecanoyl alaninate), poly(sodium N-undecanoyl leucinate), and poly(sodium N-undecanoyl valinate) (poly(L-SUV)) were synthesized. The retention behavior of the three binaphthyl derivatives under optimum electrophoretic conditions using a single chiral additive (PS or CD) is discussed. In addition, the effect of CD cavity size and stereochemical configuration of polymeric surfactants on selectivity (alpha) and resolution (Rs) was investigated. The enantioseparation of (+/-)1,1'-binaphthyl-2,2'-diamine gave a reversal of enantiomeric order when using beta-CD in combination with any of the three D-configuration PS. However, better enantioseparation is obtained when using the corresponding L-configuration PS with beta-CD. A reversal of migration order (RMO) for the enantiomers of (+/-)1,1'-bi-2-naphthol was observed upon the addition of 10 mM gamma-CD to poly(L-SUV). However, no RMO of (+/-)1,1'-bi-2-naphthol was seen when either beta-CD or gamma-CD was combined with D-PS. The enantiomers of (+/-)1,1'-binaphthyl-2,2'-diyl hydrogen phosphate showed little enantioselective behavior toward the PS alone. However, combined D- or L-PS and beta-CD or gamma-CD systems gave increased Rs and alpha values. The chiral recognition of binaphthyl derivatives observed resulting from the various combinations of two chiral selectors is discussed.

L-leucine methyl ester: the female-produced sex pheromone of the scarab beetle, Phyllophaga lanceolata.

The female-produced sex pheromone of the scarab beetle Phyllophaga lanceolata was identified as the methyl ester of an essential amino acid, L-leucine. During field testing, 239 male P. lanceolata were caught in traps baited with L-leucine methyl ester. L-Isoleucine and L-valine methyl esters, similar in structure to L-leucine methyl ester and previously identified as female-produced sex pheromone compounds employed by other Phyllophaga species, were also tested. Addition of L-valine or L-isoleucine methyl esters to the L-leucine methyl ester in 1:1 ratios completely inhibited attraction of P. lanceolata males. Males of P. squamipilosa were also captured using L-leucine methyl ester. This is the first record of P. squamipilosa from Kansas.

[Studies on chemical constituents of the mycelia from fermented culture of Flammulina velutipes].

OBJECTIVE: To study the chemical constituents from the mycelia of Flammulina velutipes. METHOD: The compounds were isolated with silica gel column chromatography and their structures were elucidated on the basis of spectral analysis (IR, EL-MS, FAB-MS, 1H-NMR, 13NMR). RESULT: Seven compounds were identified as cyclo-(R-pro-R-leu) (1), cyclo-(R-isoleu-R-leu) (2), phenylalanine (3), alanine (4), leucine (5), guanosine (6), adenosine (7), CONCLUSION: The compounds 1-6 were isolated from the mycelia of Flammulina velutipes for the first time.