Cutinase inhibition by means of insecticidal organophosphates and carbamates. 3. Oxidation of phosphorothionates by chloroperoxidase from Caldariomyces fumago

Chloroperoxidase (CPO) from Caldariomyces fumago combined with hydrogen peroxide and chloride proved to be most efficient for the transformation of organophosphorothionate pesticides, i.e., chlorpyrifos, chlorpyrifos-methyl, parathion, and parathion-methyl, into their more potent serine esterase inhibiting oxon analogues. Following CPO pre-oxidation steps, the detection limit of a recently described spectrophotometric cutinase assay could be increased by about 2 orders of magnitude as a consequence of increased inhibition rates of the organophosphates. This type of enzymatic oxidation is easier to perform and more efficient, as compared to bromine or N-bromosuccinimide, used for acetylcholine esterase (AChE) assay in water analyses, but is insufficient for complex matrices such as plant sample extracts. The performance of a complete assay, including sample preparation, oxidation, and inhibition, takes about 3 h. Performing oxidations of organophosphorus compounds, two significant anomalies were observed. Upon CPO oxidation, chlorpyrifos-methyl showed a very strong cutinase inhibition as compared to the corresponding oxon standard, and oxidized malathion, contrarily to malaoxon, revealed cutinase inhibition, which however obeyed a reversible reaction mechanism in contrast to the usually irreversible reactions of organophosphates. Except for methomyl, no significant effects of CPO oxidation on the inhibition strength of insecticidal carbamates could be detected. The applicability of the assay was tested with fruit samples spiked with chlorpyrifos at 0.2-0.5 mg/kg, thereby regarding the role of the latter as the pesticide detected most often in fruits. Mean recoveries ranged between 30-50%. An enhanced recovery of 84% was obtained for an apple juice sample spiked with parathion-methyl (0.5 mg/L).     

Development of pyrethroid substrates for esterases associated with pyrethroid resistance in the tobacco budworm, Heliothis virescens (F.)

Assays to detect esterases associated with resistance to organophosphorus and pyrethroid insecticides in larvae of H. virescens were developed and evaluated. Cross-resistance to a variety of insecticides was measured in strains resulting from selection with either profenofos (OP-R) or cypermethrin (PYR-R), and resistance in both strains appeared to have a metabolic component. Esters were synthesized that coupled 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate, the acid moiety of some pyrethroid insecticides, with groups (e.g., p-nitrophenyl-) that could be detected spectrophotometrically following hydrolysis of the resulting esters. Activities toward these pyrethroid esters were significantly higher in both resistant strains than those in a susceptible reference strain. In addition, all pyrethroid esters significantly increased the toxicity of cypermethrin in bioassays with larvae from both PYR-R and OP-R strains. The biological and biochemical activities of these compounds are compared with those with more conventional esterase substrates and insecticide synergists, and the utility of pyrethroid esters as components of rapid assays for detecting esterases associated with insecticide resistance is discussed.     

Multiresidue screen for organophosphorus insecticides using gel permeation chromatography--silica gel cleanup.

A multiresidue screen for quantitative determination of 43 organophosphorus insecticides in 5 g of plant and animal tissues is described. The organophosphorus insecticides are extracted with methanol-dichloromethane (10 + 90, v/v) and cleaned up using automated gel permeation chromatography with hexane-ethyl acetate (60 + 40) eluant and in-line silica gel minicolumns. Concentrated extracts are analyzed by gas chromatography with flame photometric detection. The method recovers 43 organophosphorus insecticides in the range of 72 to 115%. Analysis of fortified bovine liver (n = 5) shows an average 95.9 +/- 4.8% recovery at the 0.05 micrograms/g level and 93 +/- 3.8% at the 0.5 micrograms/g level. Analysis of fortified bovine rumen content (n = 5) shows an average 98 +/- 4.2% recovery at the 0.1 micrograms/g level and 98.7 +/- 2.8% at the 1 micrograms/g level. Method detection limits ranged from 0.01 to 0.05 micrograms/g for the compounds studied using a nominal 5 gram sample.     

Inhibition of soil urease by organophosphorus insecticides

The effect of three organophosphorus insecticides on soil urease was examined. Inhibition of urea hydrolysis, some 60 days after application of 1000 parts/10⁶ of insecticide to a sandy clay loam, approached 40% (accothion) and exceeded 50% in the case of malathion and thimet. Similar inhibitory effects were recorded using a silt loam soil with which 200 parts/10⁶ application also produced inhibition ranging from 14% (accothion) to 23% (thimet) after 10 days. With lower concentrations of insecticide (50 parts/10⁶) inhibition, though again significant, was of a more ephemeral nature.All three insecticides, at a concentration of 1000 parts/10⁺⁶, prevented almost any hydrolysis of urea by jack bean urease. Ureolytic microorganisms, isolated from the soils under investigation, were inhibited by the organophosphates to a greater or lesser extent but the development of tolerance was common.It is suggested that the application of insecticides to control soil-borne insect pests may be a factor in determining the efficiency of urea fertilizer mineralization.     

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.     

Comparative toxicity of selected organophosphate insecticides against resistant and susceptible clones of the greenbug, Schizaphis graminum (Homoptera: aphididae)

Comparative toxicity of selected organophosphate (OP) insecticides against resistant and susceptible clones of the greenbug, Schizaphis graminum, were studied both in vitro and in vivo. Two resistant (OR-1 and OR-2) clones of the greenbug showed marginal to high levels of resistance to all seven OPs tested, ranging from 11- to 327-fold greater than those of a susceptible (OSS) clone. The OR-1 clone showed lower levels of resistance to phenyl (parathion and parathion-methyl) and heterocyclic (chlorpyrifos) OPs than to aliphatic OPs (dimethoate, omethoate, disulfoton, and demeton-S-methyl), whereas the OR-2 clone showed a rather broad spectrum of resistance to nearly all OP insecticides examined. In vitro inhibition of acetylcholinesterase (AChE) using six selected OP oxon analogues showed that alterations of AChE were involved in resistance to all OP compounds examined in both the OR-1 and OR-2 clones. Although the levels of insensitivity of AChE to these OPs were relatively low, ranging from 1.1- to 3.8-fold, the insensitivity spectrum of AChE to different OPs was rather broad. The general esterase activity in the OR-1 and OR-2 clones was 1.3-8. 4-fold higher than that in the OSS clone, depending on the substrates used. The AChE activity in both the OR-1 and OR-2 clones was 1.8-fold higher than that in the OSS clone. High resistance levels of the OR-2 clone to phenyl and heterocyclic OPs appeared to be associated with the ability of the esterases to hydrolyze beta-naphthyl acetate and more hydrophobic substrates.     

Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives (haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer, an antigen-coated ELISA was developed, which showed an IC(50) of 3.9 ng/mL with a detection limit of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC(50) of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophos-ethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.     

Direct determination of p-nitrophenyl substituent organophosphorus nerve agents using a recombinant Pseudomonas putida JS444-modified Clark oxygen electrode

A microbial biosensor for rapid, sensitive, selective, and cost-effective determination of the total content of organophosphorus nerve agents with p-nitrophenyl substituent is reported. The biosensor consisted of genetically engineered PNP-degrader Pseudomonas putida JS444 expressing organophosphorus hydrolase (OPH) on its cell surface immobilized on a dissolved oxygen electrode. Surface-expressed OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, methyl parathion, and parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Pseudomonas putida JS444 to carbon dioxide while consuming oxygen. The oxygen consumption was measured and correlated to the concentration of organophosphates. The sensor signal and response time were optimized with 0.086 mg dry weight of cell and operating in 50 mM pH 7.5 citrate-phosphate buffer with 50 microM CoCl(2) at room temperature. When operated at optimized conditions, the biosensor measured as low as 55 ppb of paraoxon, 53 ppb of methyl parathion, and 58 ppb of parathion without interference from most phenolic compounds and other commonly used pesticides, such as atrazine, coumaphos, sutan, sevin, and diazinon. The operational life of the microbial biosensor was approximately 5 days when stored in the operating buffer at 4 degrees C.     

Self-diffusion coefficient of water in tofu determined by pulsed field gradient nuclear magnetic resonance

The self-diffusion coefficient of water in soybean protein dispersion and tofu was measured by pulsed field gradient (PFG) NMR. A soy protein isolate (SPI) dispersion (6 and 12%, w/w) in water, calcium cross-linked precipitate, and tofu were used for comparison. The self-diffusion coefficient of water (D) in the SPI dispersion, 2.23 x 10(-9) m2/s, was estimated lower than that of free water, 2.6 x 10(-9) m2/s at 25 degrees C, and decreased as the SPI concentration increased. It further decreased by the addition of calcium chloride, reflecting the obstruction effect induced by the precipitates in addition to the hydration and hydrodynamic interaction in the protein dispersion. The two water regions in tofu were interpreted by the two-site K?rger model: D1 and D2 of soft tofu were 2.26 (+/-0.11) x 10(-9) and 6.84 (+/-0.34) x 10(-11) m2/s, respectively. The relative amount of proton (water) was p1 = 0.98 and p2 = 0.02 at 100 ms of diffusion time. The self-diffusion coefficients of water decreased in pressed tofu, and their relative amounts of water changed to p1 = 0.93 and p2 = 0.07. It was suggested that D1 corresponded to obstructed water in the network structure and D2 corresponded to hydrated water on the surface layer of pores formed in the protein network of tofu. The pore sizes estimated from the diffusion length of obstructed water were 21.3 microm in soft tofu and 20.8 microm in pressed tofu. The removal of fat from pressed tofu led to a decrease in D2 from 6.26 (+/-0.31) x 10(-11) to 3.53 (+/-0.18) x 10(-11) m2/s, and the relative amount of hydrated water increased from 0.07 to 0.14, which indicated hydrophobic hydration.     

Matrix solid phase dispersion (MSPD) extraction and gas chromatographic screening of nine chlorinated pesticides in beef fat

A multiresidue technique is presented for the extraction and quantitative gas chromatographic screening of 9 insecticides (lindane, heptachlor, aldrin, heptachlor epoxide, p,p'-DDE, dieldrin, endrin, p,p'-TDE, and p,p'-DDT) as residues in beef fat. Beef fat was fortified by adding the 9 insecticides, plus dibutyl chlorendate as internal standard, to 0.5 g portions of beef fat and blending with 2 g C18 (octadecylsilyl)-derivatized silica. The C18/fat matrix blend was fashioned into a column by adding the blend to a 10 mL syringe barrel containing 2 g activated Florisil. The insecticides were then eluted from the column with 8 mL acetonitrile, and a 2 microL portion of the acetonitrile eluate was then directly analyzed by gas chromatography with electron capture detection. Unfortified blank controls were treated similarly. The acetonitrile eluate contained all of the pesticide analytes (31.25-500 ng/g) and was free of interfering co-extractants. Correlation coefficients for the 9 extracted pesticide standard curves (linear regression analysis, n = 5) ranged from 0.9969 (+/- 0.0021) to 0.9999 (+/- 0.0001). Average relative percentage recoveries (85 +/- 3.4% to 102 +/- 5.0%, n = 25 for each insecticide), inter-assay variability (6.0 +/- 1.0% to 14.0 +/- 6.7%, n = 25 for each insecticide), and intra-assay variability (2.5-5.1% n = 5 for each insecticide) indicated that the methodology is acceptable for the extraction, determination, and screening of these residues in beef fat.     

Multiresidue matrix solid phase dispersion (MSPD) extraction and gas chromatographic screening of nine chlorinated pesticides in catfish (Ictalurus punctatus) muscle tissue

A multiresidue technique for extraction and gas chromatographic screening of 9 insecticide (lindane, heptachlor, aldrin, heptachlor epoxide, p,p'-DDE, dieldrin, endrin, p,p'-TDE, and p,p'-DDT) residues in catfish (Ictalurus punctatus) muscle tissue is presented. The 9 insecticides, plus dibutyl chlorendate internal standard, were fortified into catfish muscle tissue (0.5 g) and blended with 2 g C18 (octadecylsilyl derivatized silica reverse-phase material). The C18/muscle tissue matrix blend was fashioned into a column by adding the blend to a 10 mL syringe barrel containing 2 g activated Florisil. The insecticides were then eluted from the column with acetonitrile (8 mL), and a portion (2 microL) of the acetonitrile eluate was then directly analyzed by gas chromatography with electron capture detection. Unfortified blank controls were treated similarly. The resultant extracts contained pesticide analytes (31.25-500 ng/g) free of interfering compounds when analyzed. Correlation coefficients for the 9 extracted pesticide standard curves (linear regression analysis, n = 5) ranged from 0.9967 (+/- 0.0018) to 0.9999 (+/- 0.0001). Average percentage recoveries (82 +/- 4.8% to 97 +/- 3.6%, n = 25 for each insecticide), interassay (5.0 +/- 2.7% to 16.9 +/- 6.5%, n = 25 for each insecticide) and intraassay (1.8 to 4.7%, n = 5 for each insecticide) variabilities were indicative of an acceptable methodology for the analysis and screening of these residues in catfish muscle tissue.     

Organophosphate inhibition of an arylesterase from Lumbricus terrestris and reversal by pralidoxime

Several enzymes of xenobiotic metabolism have been identified in soil annelids. Their roles in the physiology and ecotoxicology of the organism are diverse and may parallel those found in mammalian species. Among these are the arylesterases. An arylesterase assayed by hydrolysis of p-nitrophenylacetate was identified in Lumbricus terrestris. This arylesterase appears to be a serine esterase, since it is sensitive to inhibition by metrifonate. This mechanism of inhibition is similar to that of cholinesterase, since pralidoxime can reverse the inhibition of arylesterase by metrifonate. The presence of this esterase in soil annelids may provide insight into the processing of environmental chemicals by soil annelids as well as in vitro commercial applications of esterases based upon their mechanism of catalysis and inhibition.     

Barley Contains Two Cationic Acetylxylan Esterases and One Anionic Feruloyl Esterase

Various carbohydrate‐active esterases are detected in extracts of malted barley when analyzed by polyacrylamide gel electophoresis. The slowest migrating and most heat‐resistant of these are relatively cationic acetylxylan esterases. Two such activities, one with a high affinity for esterase substrates including acetylated xylan, and one with a low affinity, are indicated. These enzymes did not hydrolyze methyl ferulate. A relatively heat‐labile anionic feruloyl esterase has also been purified. It has some, albeit low, ability to act on acetylated xylan. The feruloyl esterase effects extensive release of ferulate from endosperm cell walls isolated from barley, whereas the acetylxylan esterases are only capable of very limited release of acetate.     

Crocetin, dimethylcrocetin, and safranal bind human serum albumin: stability and antioxidative properties

Crocetin (CRT) and dimethylcrocetin (DMCRT) are derived from crocins which are found in the stigmas of saffron (Crocus sativus L.), while safranal is the main component of saffron's essential oil. The aim of the present study was to examine their interaction with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various ligand contents. FT-IR and UV-visible spectroscopic methods were used to determine the ligands' binding mode, the binding constant, and the effects of ligand complexation on protein secondary structure. Structural analysis showed that crocetin, dimethylcrocetin, and safranal bind nonspecifically (H-bonding) via protein polar groups with binding constants of Kcrt =2.05 (+/-0.30) x 103 M-1, Kdmcrt = 9.60 (+/-0.35) x 104 M-1, and Ksaf = 2.11 (+/-0.35) x 103 M-1. The protein secondary structure showed no major alterations at low ligand concentrations (1 microM), whereas at higher content (1 mM), decrease of alpha-helix from 55% (free HSA) to 43-45% and increase of beta-sheet from 17% (free HSA) to 18-22% and random coil 7% (free HSA) to 10-14% occurred in the ligand-HSA complexes. The results point to a partial unfolding of protein secondary structure at high ligand content. The antioxidant activity of CRT, DMCRT, and safranal was also tested by the DPPH* antioxidant activity assay, and their IC50 values were compared to that of well-known antioxidants such as Trolox and butylated hydroxy toluene (BHT). The IC50 values of CRT and safranal were 17.8 +/- 1 microg/mL and 95 +/- 1 microg/mL, respectively, while the inhibition of DMCRT reached a point of 38.8%, which corresponds to a concentration of 40 microg/mL, and then started to decrease. The IC50 values of Trolox and BHT were 5.2 +/- 1 microg/mL and 5.3 +/- 1 microg/mL, respectively.     

Protective effects of bilberry (Vaccinium myrtillus L.) extract on restraint stress-induced liver damage in mice

Our experiments showed that 18 h restraint stress could induce serious liver damage, with an increase in plasma alanine aminotransferase (ALT) level (107.68 +/- 3.19 U/L vs 18.08 +/- 1.46 U/L). Meanwhile, we observed increased malondialdehyde (MDA) levels and lowered oxygen radical absorbance capacity (ORAC) values in plasma and liver of restraint mice compared with starved mice. Bilberry extract (containing 42.04% anthocyanins) was oral administrated to mice at 50, 100, and 200 mg/(kg x day) for five days, which remarkably decreased plasma ALT level to 17.23 +/- 2.49 U/L at the dose of 200 mg/(kg x day) and thus alleviated stress-induced liver damage. In addition, bilberry extracts increased glutathione (GSH) and vitamin C levels and significantly decreased MDA and nitric oxide (NO) levels in the liver tissues. These results suggest that bilberry extract plays an important role in protecting against restraint stress-induced liver damage by both scavenging free radicals activity and lipid peroxidation inhibitory effect. This study showed the beneficial health effects of bilberry extract through its antioxidative action.     

Degradation of Prototype Pesticides Submitted to Conventional Water Treatment Conditions: The Influence of Major Parameters

The behavior of several pesticides in aqueous solution, namely bifenthrin, amethrin (pyrethroid insecticides), endosulfan and endosulfan sulfate (organochlorine pesticides), disulfoton, methyl pyrimiphos, and phorate (organophosphorus pesticides), submitted to the conditions typically employed in water treatment stations was investigated. Continuous pesticide depletion was monitored by solid-phase microextraction sampling followed by gas chromatography–mass spectrometry analysis. The influence of major parameters (sodium hypochloride concentration, solution pH, and exposure time to ultraviolet (UV) light) was, thus, adequately established via two complementary approaches: factorial (2³, three variables—two levels) and Doehlert designs. Hence, the sodium hypochloride concentration and the solution pH produced distinct effects depending on the pesticide evaluated (for instance, acidic and basic media caused increasing rates of degradation for the organophosphorus/pyrethroid and organochlorine pesticides, respectively). Conversely, higher rates of degradation were achieved for all of the pesticides investigated when increased exposure times to UV radiation were employed. Finally, the exposure time to UV radiation that lead to complete degradation of disulfoton and endosulfan sulfate (organophosphorus and organochlorine pesticides, respectively) in aqueous media under ordinary conditions employed in water treatment stations was established; disulfoton and endosulfan sulfate were completely degraded after 10 and 40 h, respectively.     

Evaluation of chiral alpha-cyanoesters as general fluorescent substrates for screening enantioselective esterases

Esterases play a crucial role in industrial chemical synthesis, maintaining normal physiological metabolism and detoxifying exogenous ester-containing toxicants. To meet the rapidly increasing industrial need for all kinds of esterases, especially enantioselective esterases used to generate highly pure chiral compounds, general substrates are necessary for rapid screening, monitoring, purification, and characterization. In this study, general fluorescent substrates including phenolic derivatives and alpha-cyanoesters were evaluated for sensitivity in detecting esterases in buffer systems. Results with two different esterases and different incubation times suggested that the alpha-cyanoesters examined were significantly more sensitive at detecting esterases than the corresponding tested phenolic derivatives. More importantly, alpha-cyanoesters, containing a secondary alcohol, possess at least one chiral center; thus, they are tools to screen for enantioselective hydrolysis. Results indicated that the enantioselectivity of esterases toward general alpha-cyanoesters strongly depended on the esterase and the substrate, but the majority of esterases examined preferred S-isomers to their corresponding R-enantiomers. Most appealing was the very high enantioselectivity displayed in cytosolic esterases of the house fly. The potential utility of such esterases is discussed. In addition, the use of alpha-cyanoesters as chiral fluorescent substrates was demonstrated for monitoring in enantioselective esterases.     

Inhibitory effect of alpha-glucosidase inhibitors varies according to its origin

The inhibitory effect of alpha-glucosidase (AGH) inhibitors against its origins (baker's yeast and rat, rabbit, and pig small intestines) was investigated. All inhibitors used in this study showed quite different inhibitory activities according to AGH origins. Voglibose, acarbose and glucono-1,5-lactone strongly inhibited mammalian AGHs, whereas no or less inhibition was observed in yeast AGH. On the contrary, (+)-catechin, a good inhibitor against yeast AGH (IC(50) = 1.3 x 10(-)(1) mM) as well as voglibose (IC(50) = 2.6 x 10(-)(2) mM), did not retard the mammalian AGH activity. Subsequent inhibition study with various food components revealed that all of foods except for green (IC(50) = 0.735 mg/mL) and oolong teas (IC(50) = 1.34 mg/mL) showed no inhibitory activity against rat AGH, whereas they inhibited yeast AGH. Consequently, the magnitude of AGH inhibition was greatly affected by its origin, and more attention relating to AGH origin would be needed to evaluate in vitro AGH inhibitory effect.     

Determination of carbadox-related residues in swine liver by gas chromatography/mass spectrometry with ion trap detection

The ion trap detector (ITD), in combination with a capillary gas chromatograph and under chemical ionization conditions, offers sufficient sensitivity to determine carbadox-related residues as the methyl ester derivative of quinoxaline-2-carboxylic acid at 3 micrograms/kg or higher in porcine liver. A tetradeuterated internal standard of QME effectively compensates for losses incurred during sample preparation. The method produced mean levels of 3.3 (+/- 0.5), 5.5 (+/- 0.8), and 10.1 (+/- 0.9) micrograms/kg for liver fortified at 3, 5, and 10 micrograms/kg. When applied to analysis of samples containing incurred residues of 14C-carbadox at the low microgram/kg level, results were comparable to those obtained by reverse isotope dilution analysis.     

Zinc chloride-diphenylamine reagent for thin layer chromatographic detection of some organophosphorus and carbamate insecticides

Zinc chloride-diphenylamine reagent, whose use has been reported for the detection of organochlorine insecticides by thin layer chromatography, was further studied for its ability to detect the organophosphorus insecticides phorate, phosphamidon, DDVP, and phosalone and the carbamate insecticide carbaryl and aldicarb. These insecticides give intense blue-green spots with this reagent. The procedure can be applied to the detection of the insecticides in biological materials and thus has a potential use in forensic toxicology.