A method to assess glyphosate, glufosinate and aminomethylphosphonic acid in soil and earthworms.

A new sensitive and selective analytical methodology to quantify glyphosate (GLY), aminomethylphosphonic acid (AMPA), and glufosinate (GLU) in both soil and earthworms (Allolobophora chlorotica) was developed. The extraction and purification methods were optimized. The samples were extracted with various aqueous solutions (HNO3, H2O, KOH and borate buffer) and derivatized with 9-Fluorenylmethyl chloroformate (FMOCCl). To optimize the extraction step, a method to remove the excess FMOCCl was applied based on liquid-liquid extraction with diethyl ether. The purification of derivatized extracts was carried out using XLB solid phase extraction (SPE) cartridges before internal standard quantification by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The elution step was optimized to obtain the best recoveries possible, which was with acidic methanol (1% formic acid) (67% for GLY, 70% for GLU and 65% for AMPA). The extraction and purification method followed by analysis of the two herbicides and AMPA in soils using LC/MS/MS determined limit of quantification (LOQ) values of 0.030 µg g - 1 for GLY, 0.025 µg g - 1 for AMPA and 0.020 µg g - 1 for GLU . For earthworms, LOQ were 0.23 µg g - 1 for GLY, 0.20 µg g - 1 for AMPA and 0.12 µg g - 1 for GLU. . The developed method was applied to determine these compounds in natural soils and earthworms.

Synchronized determination of sacubitril and valsartan with some co-administered drugs in human plasma via UPLC-MS/MS method using solid-phase extraction.

An accurate and sensitive UPLC-MS/MS method was developed and validated for the simultaneous estimation of the newly developed combination of sacubitril and valsartan and the co-administered drugs nebivolol, chlorthalidone and esomeprazole in human plasma. Solid-phase extraction was conducted for the purification and extraction of the drugs from human plasma. Chromatographic separation was carried out on an Agilent SB-C18 (1.8 µm, 2.1 × 50 mm) column using losartan as internal standard. Isocratic elution was applied using acetonitrile-0.1% formic acid in water (85: 15, v/v) as mobile phase. Detection was carried out using a triple-quadrupole tandem mass spectrometer using multiple reaction monitoring, at positive mode at m/z 412.23 → 266.19 for sacubitril, m/z 436.29 → 235.19 for valsartan, m/z 405.8 → 150.98 for nebivolol, m/z 346.09 → 198 for esomeprazole and a selected combination of two fragments m/z 423.19 → 207.14 and 423.19 → 192.2 for losartan (internal standard), and in negative ionization mode at m/z 337.02 → 190.12 for chlorthalidone. The method was linear over the concentration ranges 30-2,000 ng/ml for sacubitril, 70-2,000 ng/ml for valsartan, esomeprazole and chlorthalidone and 70-5,000 pg/ml for nebivolol. The developed method is sensitive and selective and could be applied for dose adjustment, bioavailability and drug-drug interaction studies.

One-step purification/extraction method to access glyphosate, glufosinate, and their metabolites in natural waters.

A new green method for trace level quantification of four herbicides, glyphosate (GLYP), glufosinate (GLUF), and their main metabolites, aminomethylphosphonic acid (AMPA) and 3-(methyl-phosphinico)-propionic acid (MPPA), was developed. The purification step without any derivatization was conducted by solid-phase extraction using Chelex-100 resin in the Fe (III) form, followed by elution with 5% NH4OH. The four analytes were quantified by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. The developed extraction method was validated on five fresh and sea water matrices with mean recoveries ranging from 80.1% to 109.4% (relative standard deviation < 20%). The extraction conditions were evaluated and certified for the high applicability of the extraction method too. The limits of detection (ng/L) in the five water matrices were in ranges 0.70 - 4.0, 2.4 - 3.9, 1.8 - 4.7, and 1.6 - 4.0 for GLYP, AMPA, GLUF, and MPPA, respectively. The method was successfully applied to detect the four compounds in surface waters sampled along the Red River Delta region in July 2019. The highest concentrations were detected at 565, 1,330, 234, and 871 ng/L for GLYP, AMPA, GLUF, and MPPA, respectively. These results showed the potential capacity of this new method for convenient monitoring of herbicides and their metabolites in the diverse natural water system.

1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite-Malylglutamate.

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

Simultaneous LC-MS/MS analysis of glyphosate, glufosinate, and their metabolic products in beer, barley tea, and their ingredients.

Glyphosate and glufosinate are non-selective herbicides that have been extensively used worldwide. Their ionic and water-soluble characteristics often make it difficult to analyze them, especially in food components. A method was developed in this study for the simultaneous analysis of glyphosate, glufosinate, and three metabolic products in beer, barley tea, and their ingredients (malt and corn). The analytical samples were extracted with H2O, purified with a strong anion-exchange solid-phase extraction (SPE) cartridge, and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an anion-exchange high-performance liquid chromatography (HPLC) column. This method enabled a rapid and sensitive analysis [limit of quantification (LOQ) = 10 µg/kg] of the herbicides to be achieved.

Elucidation of matrix effects and performance of solid-phase extraction for LC-MS/MS analysis of ß-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) neurotoxins in cyanobacteria.

A liquid chromatography-mass spectrometry (LC-MS/MS) method using hydrophilic interaction liquid chromatography (HILIC) was developed for the analysis of neurotoxins ß-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), using multiple reaction monitoring (MRM) scan mode. Oasis-MCX and Strata-X-C polymeric cation-exchange cartridges were used to clean extracts of cyanobacterial cultures, including two strains of Microcystis aeruginosa and one strain of Nostoc sp. The performance of the solid-phase extraction (SPE) cartridges for BMAA and DAB were evaluated using mixed standards and spiked cyanobacterial extracts, which demonstrated recoveries of BMAA and DAB ranging from 66% to 91%. Matrix effects in LC-MS/MS were evaluated, and while there was no effect on BMAA quantitation, suppression of DAB was found. Full scan (Q1) and enhanced product ion (EPI) monitoring showed that the DAB suppression may be due to closely eluting compounds, including lysine, histidine, arginine and three other compounds with [M + H](+) m/z of 88, 164 and 191. The procedures developed allow the sensitive and effective analysis of trace BMAA and DAB levels in cyanobacteria. While DAB was confirmed to be present, no BMAA was found in the cyanobacterial samples tested in the present study.

Optimization of extraction procedure and chromatographic separation of glyphosate, glufosinate and aminomethylphosphonic acid in soil.

Analysing herbicides in soil is a complex issue that needs validation and optimization of existing methods. An extraction and analysis method was developed to assess concentrations of glyphosate, glufosinate and aminomethylphophonic acid (AMPA) in field soil samples. After testing extractions by accelerated solvent extraction and ultrasonic extraction, agitation was selected with the best recoveries. Water was preferred as solvent extraction because it resulted in a cleaner chromatogram with fewer impurities than was the case with alkaline solvents. Analysis was performed by FMOC pre-column derivatization followed by high-performance liquid chromatography (HPLC) on a 300 mm C(18) column which permitted enhanced separation and sensitivity than a 250 mm C(18) column and increased resistance than the NH(2) column for soil samples. This extraction and analysis method allowing a minimum of steps before the injection in the HPLC with fluorescence detection is efficient and sensitive for a clay-loamy soil with detection limits of 103 µg kg(-1) for glyphosate, 15 µg kg(-1) for glufosinate and 16 µg kg(-1) for AMPA in soil samples.

Analytical protocol for identification of BMAA and DAB in biological samples.

Beta-N-methylamino-L-alanine (BMAA) is a non-protein amino acid, thought to be inflicting neurodegenerative diseases related to ALS/PDC in human beings. Due to conflicting data concerning the presence of BMAA in various biological matrixes, we present a robust and sensitive method for high confidence identification of BMAA after derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The efficient sample pretreatment in combination with LC-MS/MS SRM enables chromatographic separation of BMAA from the isomer 2,3-diaminobutyric acid (DAB). The method is applicable for selective BMAA/DAB detection in various biological samples ranging from a prokaryotic cyanobacterium to eukaryotic fish.

Enantioseparation of beta-methyl-substituted amino acids with cyclodextrins by capillary zone electrophoresis.

Direct capillary zone electrophoretic methods were developed for the separation of the enantiomers of unnatural beta-methyl-amino acids such as erythro- and threo-beta-methylphenylalanine, beta-methyltyrosine, beta-methyltryptophan and beta-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Capillary zone electrophoresis was carried out using sulfopropylated-alpha-CD (SP2-alpha-CD), sulfopropylated-beta-CD (SP2-beta-CD) both with a degree of substitution of 2 moles/mole cyclodextrin, and sulfopropylated-beta-CD (SP4-beta-CD) with a degree of substitution of 4moles/mole beta-cyclodextrin. The effects of selector and buffer concentrations, electrolyte pH and applied voltage were studied on the separation efficiency. Varying the electrophoretic conditions with application of 20 kV, hydrodynamic injection, unmodified silica capillary, three different buffers (borate, phosphate and acetate) and modified cyclodextrins as chiral selectors all compounds investigated are nearly baseline resolved. The elution sequence was determined in most cases.

Structure and semisynthesis of platensimide A, produced by Streptomyces platensis.

Platensimycin and platencin are novel natural product antibiotics that inhibit bacterial growth by inhibiting condensing enzymes FabF and FabF/FabH of fatty acid biosynthesis pathways, respectively. Continued search for the natural congeners of these compounds led to the isolation of platensic acid, the free C-17 tetracyclic enoic acid, and platensimide A, a 2,4-diaminobutyric acid amide derivative. Isolation, structure, semisynthesis, and activity of these compounds are described.

Osmoprotection of Salmonella enterica serovar Typhimurium by Ngamma-acetyldiaminobutyrate, the precursor of the compatible solute ectoine.

N(gamma)-acetyl-2,4-diaminobutyrate (NADA), the precursor of the compatible solute ectoine, was shown to function as an osmoprotectant for the non-halophilic bacterium Salmonella enterica serovar Typhimurium. The addition of NADA-containing extracts of an ectoine synthase mutant of the broad salt-growing halophile Chromohalobacter salexigens DSM 3043(T) could alleviate the inhibitory effects of high salinity in S. enterica, which lacks the ectoine biosynthetic pathway. NADA, purified from extracts of the mutant, protected S. enterica against salinity stress. This osmoprotective effect was slightly lower than that of ectoine, but more potent than that of hydroxyectoine. Accumulation of purified NADA by S. enterica was demonstrated by (13)C-NMR spectroscopy and HPLC analysis. In addition, it was shown that NADA was taken up by S. enterica via the ProP and ProU transport systems, which are known to transport glycine betaine and proline. This finding provides evidence that these permeases can recognize a diaminoacid that carries an unsubstituted alpha-amino group. This is the first time that NADA has been connected with osmoprotective functions in non-halophilic bacteria.

Preparative separation and identification of derivatized beta-methylphenylalanine enantiomers by chiral SFC, HPLC and NMR for development of new peptide ligand mimetics in drug discovery.

A direct preparative purification of all four isomers of the unnatural amino acid beta-methylphenylalanine was achieved using supercritical fluid chromatography (SFC) with stacked-injection. Final purification of the Cbz-methyl ester derived isomers was performed on a Daicel Chiralpak AD-H column (20 mm x 250 mm), using 50:50 methanol/ethanol as the organic modifier and resulted in purification of over 3.4 g of material in 6.25 h with >90% total recovery. The absolute stereochemical assignment of the purified amino acids was determined through a combination of chiral HPLC, NMR and optical rotation studies. To our knowledge, this is the first reported preparative approach that has yielded all four compounds in a single chromatographic run.

Absolute determination method using chirality for glufosinate and bialaphos by gamma-cyclodextrin modified capillary zone electrophoresis.

A novel absolute determination method using chirality without any calibration curves or comparison standards has been proposed for phosphorus-containing amino acid-type herbicides, glufosinate (D,L-GLUF) and bialaphos (BIAL). This method is based on a change in the enantiomeric ratio after the spiking of a known amount of the enantiomers with different enantiomeric ratios to a sample. D,L-GLUF was determined by adding a known amount of L-GLUF to the sample, derivatizing them with dansyl chloride, and measuring the ratio of the peak area of the D-isomer to that of the L-isomer by means of gamma-cyclodextrin modified capillary zone electrophoresis. The accuracy and precision of the method were evaluated using a synthetic sample. The mean values obtained for D- and L-GLUF agreed with the values taken within 1.6%; also the reproducibility was as good as less than 2.8%. The determination of BIAL was achieved by determining GLUF quantitatively produced by the acid hydrolysis of BIAL. The proposed methods were applied to the analysis of commercial herbicides and the validity and usefulness were evaluated.

Analysis of phosphorus herbicides by ion-pairing reversed-phase liquid chromatography coupled to inductively coupled plasma mass spectrometry with octapole reaction cell.

A reversed phase ion-pairing high performance liquid chromatographic (RPIP-HPLC) method is developed for the separation of two phosphorus herbicides, Glufosinate and Glyphosate as well as Aminomethylphosphonic acid (AMPA), the major metabolite of Glyphosate. Tetrabutylammonium hydroxide is used as the ion-pairing reagent in conjunction with an ammonium acetate/acetic acid buffering system at pH 4.7. An inductively coupled plasma mass spectrometer (ICP-MS) is coupled to the chromatographic system to detect the herbicides at m/z = 31P. Historically, phosphorus has been recognized as one of the elements difficult to analyze in argon plasma. This is due to its relatively high ionization potential (10.5 eV) as well as the inherent presence of the polyatomic interferences 14N16O1H+ and 15N16O+ overlapping its only isotope at m/z = 31. An octapole reaction cell is utilized to minimize the isobaric polyatomic interferences and to obtain the highest signal-to-background ratio. Detection limits were found to be in the low ppt range (25-32 ng/l). The developed method is successfully applied to the analysis of water samples collected from the Ohio River and spiked with a standard compounds at a level of 20 microg/l.

Isomers of substituted 3-benzo[b]furyl and 3-thienylaminobutyric acids as potent ligands of the GABA-B receptor: synthesis and preparative liquid chromatographic separation.

Baclofen (4-amino-3-(4-chlorophenyl)butyric acid) is the only selective agonist for GABA-B receptors. Its R-(-)-enantiomer is about 100 times more active than the S-(+)-enantiomer. In the search for new compounds that bind to GABA-B receptors, it is very important to clarify the structural requirements. The authors report the synthesis and separation of isomers of various 3-heteroaromatic (benzo[b]furan and thiophen) aminobutyric acids. The 4-amino-3-(7-methylbenzo[b]furan-2-yl)butanoic acid is a potent and specific ligand for GABA-B receptors, with an IC50 value of 5.4 microM for the displacement of [3H] GABA.

Separation of enantiomeric beta-methyl amino acids and of beta-methyl amino acid containing peptides.

Erythro-D,L- and threo-D,L-beta-methylphenylalanine, -beta-methyltyrosine and -beta-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid were synthesized. High-performance liquid chromatographic methods were developed for the separation and identification of the enantiomers of the beta-methyl amino acids, with the application of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate as derivatizing reagents. These amino acids were incorporated into the mu-agonist/delta-antagonist opioid peptides H-beta-MeTyr-Tic-Phe-Phe-NH2, H-Tyr-Tic-beta-MePhe-Phe-NH2 and H-Tyr-Tic-Phe-beta-MePhe-NH2, and the delta-antagonist H-Tyr-beta-MeTic-Phe-Phe-OH, by solid-phase peptide synthesis. Each peptide has four stereoisomers. The peptide stereoisomers were separated on different columns and in different eluent systems and the elution order of the peptide epimers was determined.

Structure of a new peptide, astin J, from Aster tataricus.

A new peptide called astin J (1) was isolated from the roots of Aster tataricus. The structure was elucidated by spectroscopic methods and chemical transformation from a cyclic pentapeptide, astin C, isolated from A. tataricus to the analogous acyclic peptide, 2. Antileukemic activities of the series of acyclic peptides are also described.

Capillary zone electrophoresis study of naphthylethylcarbamoylated beta-cyclodextrins.

Regiospecifically monosubstituted 1-(1-naphthyl)ethylcarbamoylated beta-cyclodextrins (NEC-beta-CDs) were successfully employed as chiral additives in capillary zone electrophoresis (CZE) to achieve chiral separation of N-(3,5-dinitrobenzoyl)phenylglycine (3,5-DNB-PG), phenylalanine (3,5-DNB-PA), and homophenylalanine (3,5-DNB-HPA). The enantioselectivity of the various site-substituted NEC-beta-CDs in CZE was compared with that of native beta-CD. Complexation constants of the three 3,5-DNB amino acids with beta-CD were determined from the CZE results: 3,5-DNB-L-HPA, 473 +/- 9 M-1; 3,5-DNB-D-HPA, 460 +/- 10 M-1; 3,5-DNB-L-PA, 260 +/- 4 M-1; 3,5-DNB-D-PA, 161 +/- 3 M-1; and 3,5-DNB-D, L-PG, 43 +/- 4 M-1.

Antimutagenic effects of N-methyl-valyl-amiclenomycin (BA-2) isolated from the metabolites of Streptomyces sp.

A novel antimutagenic factor, BA-2, active against UV-induced mutagenesis in Escherichia coli WP2 was isolated from the metabolites of Streptomyces sp. strain AJ9455. BA-2 also suppressed mutations induced by 4-nitroquinoline N-oxide (4-NQO) and furylfuramide (AF-2) in E. coli WP2s (uvrA) without any decrease of cellular viability. BA-2 strongly inhibited the UV induction of SOS repair functions when it was monitored by beta-galactosidase activity expressed from the sulA::lacZ fusion gene of strain PQ37. It is assumed that the antimutagenic effect of BA-2 on mutagenesis induced by UV, 4-NQO or AF-2 was the result of inhibition of induction of the inducible error-prone SOS repair. The structure of BA-2 was considered to be N-methyl-valyl-amiclenomycin, and the structural unit of 4-amino-2,5-cyclohexadiene must be essential for the antimutagenic activity, since deamination by heating results in the loss of antimutagenic activity of BA-2.