Biosensor for direct determination of fenitrothion and EPN using recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> JS444 with surface-expressed organophosphorous hydrolase. 2. Modified carbon paste electrode

A whole cell-based amperometric biosensor for highly selective, sensitive, rapid, and cost-effective determination of the organophosphate pesticides fenitrothion and ethyl p-nitrophenol thiobenzenephosphonate (EPN) is discussed. The biosensor comprised genetically engineered p-nitrophenol (PNP)-degrading bacteria Pseudomonas putida JS444 anchoring and displaying organophosphorous hydrolase (OPH) on its cell surface as biological sensing element and carbon paste electrode as the amperometric transducer. Surface-expressed OPH catalyzed the hydrolysis of organophosphorous pesticides such as fenitrothion and EPN to release PNP and 3-methyl-4-nitrophenol, respectively, which were subsequently degraded by the enzymatic machinery of P. putida JS444 through electrochemically active intermediates to the TCA cycle. The electrooxidization current of the intermediates was measured and correlated to the concentration of organophosphates. Operating at optimum conditions, 0.086 mg dry wt of cell operating at 600 mV of applied potential (vs Ag/AgCl reference) in 50 mM citratephosphate buffer, pH 7.5, with 50 μM CoCl₂ at room temperature, the biosensor measured as low as 1.4 ppb of fenitrothion and 1.6 ppb of EPN. There was no interference from phenolic compounds, carbamate pesticides, triazine herbicides, or organophosphate pesticides without nitrophenyl substituent. The service life of the biosensor and the applicability to lake water were also demonstrated.     

A Practical Synthesis of 2,4-Dichloro-3-methyl-6-nitrophenol

2,4-Dichloro-3-methyl-6-nitrophenol 2 was prepared by KOH/H₂O hydrolysis of a product mixture obtained from chlorination of p-nitrotoluene in the presence of a phase transfer catalyst. A 95–99% yield of 2 based on 2,3,6-trichloro-4-nitrotoluene, 4 (major chlorination product) was achieved in >95% purity.     

Quantification of fenitrothion and its main metabolites in poplar leaves by isotope dilution gas chromatography-mass spectrometry coupled with solid-phase microextraction

A sensitive and specific method for determining fenitrothion and its main metabolites, 3-methyl-4-nitrophenol and fenitrooxon, in poplar leaves using deuterated isotopes as the internal standard is described. The analytes and the labeled isotopes were extracted from leaves by solid-phase microextraction and subsequently analysed by gas chromatography coupled with mass spectrometry. The method had a chromatographic run time of 17.0 min and good linearity over the range 0.01-10 mg kg(-1). The detection limits ranged between 2.5 and 0.6 microg kg(-1). The isotopic dilution technique allowed improving significantly the repetitivity even using different fibers with the same coating (RSD<5.1%). The method was applied successfully to study the persistence of fenitrothion in forestal matrices in a poplar forest after cannon spray application of the insecticide.     

Optimization and validation of a method of analysis for fenitrothion and its main metabolites in forestry air samples using sorbent tubes with thermal desorption cold trap injection and gas chromatography-mass spectrometry

An analytical methodology using thermal-desorption cold trap (TCT) and GC-MS was developed for the determination of the insecticide fenitrothion and its main metabolites, 3-methyl-4-nitrophenol and fenitrooxon, in forestry atmospheres. The sampled atmosphere was pumped through a glass tube containing 100 mg of Tenax adsorbent at a flow rate of 50 ml min(-1). Adsorption/thermal desorption and breakthrough experiments were performed to test the ability to quantitatively trap the compounds. The detection limits of method for these compounds ranged between 1.6 and 2.1 ng m(-3). This methodology was developed to evaluate the persistence of fenitrothion in forest atmospheres after treatment. Spray application at 21.5 mg active ingredient m(-2) resulted in atmosphere levels of the insecticide of 78.3 ng m(-3) (after 2 h of application). Within 2-4 days following treatment, the presence of fenitrooxon fell to 50-55%. During this period residues of metabolites began to appear, disappearing 19 days later.     

Solid-phase microextraction coupled with high performance liquid chromatography using on-line diode-array and electrochemical detection for the determination of fenitrothion and its main metabolites in environmental water samples

The aim of this study was to develop a methodology for the analysis of the insecticide fenitrothion and its two main environmental metabolites, fenitrooxon and 3-methyl-4-nitrophenol. For this purpose, a solid-phase microextraction (SPME) method coupled to high performance liquid chromatography (LC) was optimized. Two on-line detectors, diode array (DAD) and direct current amperometrical (DCAD) were used in order to determine sensitivity and selectivity. The effects of the extraction parameters, including exposure and desorption time, pH, temperature, salt concentration and desorption mode on the extraction efficiency were studied. A satisfactory reproducibility for extractions from samples at 20 ppb-level with RSD < 12.5% (n = 10) was obtained. The calibration graphs were linear in the range of 10-1000 microg l(-1) and detection limits for the target compounds were between 1.2 and 11.8 microg l(-1) depending on which detector was used. The method was applied for determining fenitrothion and both its metabolites in river waters which run through forest areas near to aerial application of the pesticide.     

Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/electrospray tandem mass spectrometry

The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub-ppb level is explored. Metabolites from two organophosphorous pesticides, 4-nitrophenol (from parathion and parathion-methyl) and 3-methyl-4-nitrophenol (from fenitrothion), are taken as model analytes to conduct this study. After direct injection of the urine sample (10 microL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope-labeled internal standards. The advantages of applying coupled-column liquid chromatography (LC/LC) as an efficient clean-up without any type of sample manipulation are also discussed. The combination of LC/LC with ESI-MS/MS allows the direct analysis of free metabolites in urine, as the automated clean-up performed by the coupled-column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI-MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4-nitrophenol and 3-methyl-4-nitrophenol in human urine without any sample treatment.     

Reaction of Dimethyl Acetylene-Dicarobxylate With Triazinone

Abstract

The addition of dimethyl acetylenedicarboxylate (DMAD) to 6-menthy-1, 2,4-triazlne-3(2H)thione-5(4H)-one afforded 2-methoxylcarboxy-7-methyl-1,3-thiazino[3,2-b][1,2,4]triazine-4,8-dione.     

Analysis of the Sulfonylurea Herbicide Metsulfuron-Methyl and Its Metabolites in the Soil of Cereal Crops. Comparative Analytical Chemistry of the Sulfonylureas

Abstract

For the analysis of metsulfuron-methyl in the crop soils with a sensitivity limit of 0.3 μg kg^?1 dry soil, in the soil extract metsulfuron-methyl was separated from its soil metabolites and the soil impurities by repeated thin-layer chromatographies (TLC). In the cleaned soil extract, diazomethane transformed metsulfuron-methyl 1 into N,N′ -dimethyl metsulfuron-methyl 2 (methyl 2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)methylamino]carbonyl]methylamino]sulfonyl]benzoate). In the gas-liquid chromatograph with detection by electron capture (GC-EC) and in the combined gas chromatograph-mass spectrometer (GC-MS), 2 was transformed into 1-dioxy-2-N-methyl-3-keto-1,2-benzisothiazole 3 which was measured by GC-EC with confirmation by GC-MS. The metsulfuron-methyl soil metabolites 2-sulfonamido-methylbenzoate 6, 1-dioxy-3-keto-1,2-benzisothiazole (saccharin) 7 and 2-sulfonamidobenzoic acid 8 were analyzed in the soil of winter wheat crops by a procedure similar to the one for metsulfuron-methyl. After their separation and purification in the soil extracts by TLC, 7 and 8 were methylated, and analyzed as 3 in the GC-EC and GC-MS apparatus where the generated 6 was quantitatively transformed into 3; 6 was analyzed as such with the GC and GC-MS apparatus wherein it was transformed into 3. The sensitivity limit for each metabolite was 0.3 μg of equivalents of metsulfuron-methyl kg^?1dry soil. The syntheses of the analysis standards of the metsulfuron-methyl derivatives 2 and 3, and of the metsulfuron-methyl metabolites 6, 7 and 8 are described. The transformation pathways of metsulfuron-methyl and of its derivatives are different from those of the pyridine-pyrimidine sulfonylurea herbicides flupyrsulfuron-methyl and rimsulfuron. The soil analysis of a sulfonylurea -by means of one of its transformation product- needs a previous study of the chemical reactivity of the sulfonylurea. This leads to the analysis procedures for the main soil metabolites of the sulfonylurea.     

Biodegradation and bioaccumulation of fenitrothion in rat liver

The biodegradation of fenitrothion O,O-dimethyl-O-(3-methyl-4-nitro phenyl) phosphorothioate was investigated in rat liver after administration of various doses (5 mg/100 g body weight and 20 mg/100 g body weight) in acute treatment and 1 mg/100 g body weight in chronic treatment. High performance liquid chromatography of the pesticide and its metabolites formed in liver in acute treatment showed time-dependent sequential conversion of pesticide into three major metabolites within 24 h. These metabolites were separated and purified to homogenity by HPLC and characterized by IR spectroscopy as O,O-dimethyl-O-(3-methyl-4-amino phenyl) phsophorothioate (metabolite 1), O,O-dimethyl phosphorothioate (metabolite II) and O,O-dimethyl phosphate (metabolite III) in the fi rst dose (5 mg/100 g body weight). Metabolite II was found to be different in the second dose (20 mg/100 g body weight) and identified as O,O-dimethyl O-3-methyl-4-amino phenyl phosphate. The results with the fi rst dose indicated reduction of the nitro group in fenitrothion as step I followed by hydrolytic clevage of the P-O-aryl bond in metabolite I and oxidative desulphurylation of metabolite II. At higher dose (20 mg/100 g body weight) oxidative desulphurylation takes place as step II followed by hydrolysis of metabolite II. The bioaccumulation of fenitrothion within 60 days during chronic treatment showed no metabolite but continuous reduction in fenitrothion concentration, indicating excretion of pesticide and its products in urine and in faeces.     

Detection and Identification of the Herbicide Isoproturon and its Metabolites in Field Samples After a Heavy Rainfall Event

Abstract

The occurence of isoproturon [N-(4-isopropylphenyl)-N′, N′-dimethyl urea] and its metabolites was investigated in soil solution, runoff and creek water from a farm in Scheyern (Bavaria, FRG) after a heavy rainfall event following the application in spring 1994. The analytical procedure included enrichment by C 18 solid-phase extraction and reversed-phase HPLC with diode array detection. The major degradation product in most of the samples was identified as 2-hydroxy-isoproturon [N-(4-(2-hydroxyisopropyl)-phenyl)-N′. N′ dimethyl urea] using the authentic compound. The latter has been isolated from wheat cell culture medium and its structure elucidated by NMR and MS. Furthermore, the polar metabolites monodesmethyl-isoproturon [N-′ (4-Isopropylphenyl)-N′-methyl urea] and 2-hydroxy-monodesmethyl-isoproturon [N-(4-Isopropylphenyl)-N′-methyl urea] and 2-hydroxy-monodesmethyl-isoproturon [N-(4-(2-hydroxyisopropyl)-phenyl)-N′-methyl urea] were identified by comparison of their retention times and DAD-UV spectra with reference compounds.

The detection of isoproturon in soil solution down to 170 cm depth and in creek water in concentrations exceeding 4 μg/1 and also of the polar metabolites in concentrations up to 0,9 μg/1 indicated the mobility of this phenylurea herbicide and its degradation products. Therefore the metabolites should be considered in studies on the migration of isoproturon and in the ecotoxicological evaluation.     

Group-specific enzyme-linked immunosorbent assay for fenitrooxon and 3-methyl-4-nitrophenol in water samples based on a polyclonal antibody

Fenitrooxon [O,O-dimethyl-O-(4-nitro-m-tolyl)phosphate] is the major metabolite of the organophosphorus insecticide fenitrothion, and 3-methyl-4-nitrophenol is its major degradation product. In the present study, we describe the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of these compounds in water samples based on a group-specific polyclonal antiserum generated with a “bifunctional hapten”, which has two functions: the conventional function of producing an antibody against an antigen and a unique function of promoting the production of the antibodies in rabbit. For application to water samples, the influence of several factors such as organic solvent, pH, and detergent was studied. Under optimized conditions, the quantitative working range of the fenitrooxon ELISA was 0.71-27 ng ml⁻¹ with a limit of detection (LOD) of 0.32 ng ml⁻¹, and the fenitrooxon concentration giving 50% reduction of the maximum signal (IC₅₀) was 4.2 ng ml⁻¹. The quantitative working range of the 3-methyl-4-nitrophenol ELISA was 0.67-27 ng ml⁻¹ with a LOD of 0.38 ng ml⁻¹ and an IC₅₀ of 3.7 ng ml⁻¹. No significant matrix effect originating from the water sample (river water, tap water, purified water, and bottled water) was shown by addition of Tween 20 to the assay buffer. Water samples spiked with each of these compounds at 1, 5, 10, and 20 ng ml⁻¹ were directly analyzed without extraction and clean-up by the proposed ELISA. The mean recovery was 100.9%, and the mean coefficient of variation (CV) was 7.7% for the fenitrooxon ELISA and for the 3-methyl-4-nitrophenol ELISA, the mean recovery was 97.6%, and the mean CV was 7.2%. The proposed ELISA allows precise and accurate determination of these compounds in water at such low levels.     

Concise Synthesis of 3-Hydroxy-Δ1.10−12,16-cyclogibberellin 12 Dimethyl Ester—A Trachylobagibberellin Analogue

Abstract

Chemical synthesis of a trachylobagibberellin analogue - 3-hydroxy- δ^1.10?12, 16-cyclogibberellin dimethyl ester 4 from gibberellin acid GA₃ 3 was described herein. The key step is the decomposition of the tosylhydrazone 11 to construct the novel [3.2.1.0.^2.7] octane system.     

Chromatographic Purification and Identification of Polar Metabolites of Benazolin-Ethyl from Soybean

Abstract

The metabolism of benazolin-ethyl (4-chloro-2-oxobenzothiazolin-3-ylacetic acid ethyl ester), a post emergence herbicide, has been studied in soybean using (¹⁴C)-phenyl labelled compound. Preliminary studies were performed on excised soybean leaves. Following hydrolysis of the ethyl ester to benazolin acid (4-chloro-2-oxobenzothiazolin-3-ylacetic acid), extensive metabolism to polar conjugates was observed. The polar fraction from a Bligh-Dyer extraction was purified by solvent partitioning, preparative TLC and reverse phase HPLC with ion suppression. The two major metabolites were characterised by fast atom bombardment mass spectrometry with accurate mass determination as an aspartate conjugate and a malonyl-β-glucose ester of benazolin acid. Subsequent experiments were performed by spraying intact plants at growth stage V4. The major polar metabolite isolated one month after treatment was identified as the aspartate conjugate by mass spectrometry and high resolution nuclear magnetic resonance spectroscopy.     

The ipso-nitration of p-cresol

The nitration of $\text{p}$-cresol by nitric acid in aqueous sulphuric acid involves ~40% $\text{ipso}$-substitution at C^Me. The 4-methyl-4-nitrocyclohexa-2,5-dien-1-one (I) formed undergoes an acid-catalysed rearrangement to 4-methyl-2-nitrophenol.     

Photodegradation of Selected Organophosphorus Insecticides Under Sunlight in Different Natural Waters and Soils

In this work photodegradation of four organophosphorus insecticides (ethyl-parathion, methyl-parathion, fenitrothion, fenthion) in different natural waters and soils was studied under sunlight. The origin of the waters was from the region of Ioannina (underground, lake, and river water) and from Preveza (sea water) in Northern-West Greece. The soils used had different percentages of organic matter (0.9-3.5%) and their characterization were SCL, CL, and SL respectively. The photodegradation kinetics of these insecticides were followed by GC-FTD. The identification of the photodegradation by-products was made by using GC-MS. The half-lives of the organophosphorus insecticides vary from 0.4 to 35.4 days in natural waters and from 3.4 to 21.3 days in soils. The humic substances and the other components of these environmental matrices seem to influence the degradation kinetics. The use of GC-MS allowed the identification of some important photodegradation by-products such as: fenthion sulfone, fenthion sulfoxide, fenoxon, 4-methylthio-3,5-dimethyl phenol, O , O , O -triethyl phosphorothioate, paraoxon, 4-nitrophenol, aminoparathion.     

Dual Electrochemical Detection of Biogenic Amine Metabolites in Micro High-Performance Liquid Chromatography

Abstract

The dual electrochemical detectors for ordinary and micro high-performance liquid chromatography were briefly reviewed.

The electrochemical behaviors of biogenic amine metabolites were studied by cyclic semi-differential and semi-integral voltammetry with a glassy carbon working electrode. It was found that the electrochemical reactions of many biogenic amine metabolites are quasi-reversible. The dual electrochemical detector based on thin-layer electrolytic cell with two working electrodes (anode and cathode) in series configuration was tested for selective detection of biogenic amine metabolites on their electrochemical quasi-reversibility. The detector was successfully utilized for the simultaneous determination of 3, 4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid in human urine directly injected by micro high-performance liquid chromatography.     

Adsorption of diazinon and fenitothion on MCM-41 and MCM-48 mesoporous silicas from non-polar solvent

The adsorption of two common organophosphorus pesticides, diethoxy-[(2-isopropyl-6-methyl-4-pyrimidinyl)oxy]-thioxophosphorane (diazinon) and dimethoxy-(3-methyl-4-nitrophenoxy)-thioxophosphorane (fenitrothion), by MCM-41 and MCM-48 mesoporous silicas at room temperature was investigated. UVvis and IR spectroscopy, small-angle X-ray diffraction, and the specific surface area analysis (S _BET) were used to study the adsorption behavior of diazinon and fenitrothion. The results show that the MCM-41 and MCM-48 mesoporous silicas adsorb diazinon more efficiently than fenitrothion. The extraction of adsorbed materials from the adsorbents with polar solvents and subsequent analysis by ³¹P NMR showed that the adsorption of diazinon and fenitrothion on mesoporous silicas is destructive and non-destructive, respectively. Nitrogen adsorption measurements showed that the specific surface area of both silicas decreases after the adsorption of pesticides, and the larger effect is observed for diazinon. The article is published in the original.     

Simultaneous Determination of Warfarin,Sulphaquinoxaline and Fenitrothion in Wheat-Based Rodenticide Baits by High Pressure Liquid Chromatography

Abstract

The simultaneous determination of warfarin, sulphaquinoxaline and fenitrothion in wheat-based rodenticides is achieved by extracting the three components from the bait with dimethylformaaide followed by an isocratic, high-pressure liquid chromatographic separation using a reverse-phase RP-8 column and 0.005 M pentane sulphonic acid in methanol:water (60:40) as eluent. The three components are detected at 280 nm after separation. Recoveries in the concentration range investigated were fenitrothion 97.2%, warfarin 97.8% and sulphaquinoxaline 96.9%.     

THE REACTION OF ACETYLACETONE WITH DIALKYLPHOSPHITES: Synthesis of Dialkyl 1-methyl-3-oxo-1-butenyl-phosphonates

Abstract

Acetylacetone and its related compounds were transformed into the dialkyl 1-methyl-1-trimethylsiloxyl-3-oxo-butylphosphonates, which after treatment with trifluoroacetic acid were converted into the dialkyl 1-methyl-3-oxo-1-butenylphosphonates.     

Isolation and identification of three urinary metabolites of retinoic acid in the rat.

After the intraperitoneal administration of high doses of ¹⁴C- and ³H-labelled retinoic acid ( 1 ) to rats three major urinary metabolites have been isolated in microgram amounts by use of column, thin-layer and high-pressure liquid chromatography. Their structures were elucidated by mass spectroscopy and Fourier transform ¹H-NMR. spectroscopy as 2 (5-methyl-5-[2-(2,6,6-trimethyl-3-oxo-1-cyclohexen-1-yl)vinyl]-2-tetrahydrofuranone), 3 (5-[2-(6-hydroxymethyl-2,6-dimethyl-3-oxo-1-cyclohexen-1-yl)vinyl]-5-methyl-2-tetrahydrofuranone) and 4 (6-(6-hydroxymethyl-2,6-dimethyl-3-oxo-1-cyclohexen-1-yl)-4-methyl-4-hexenoic acid). In these metabolites the tetraene side chain of 1 is shortened and the cyclohexene ring oxidized. The radioactivity of 2 and 3 accounted for about 10% (0.9% of the dose) each, metabolite 4 for about 6% (0.5% of the dose) of the total urinary radioactivity.