Synthesis and Application of Novel Molecularly Imprinted Solid Phase Extraction Materials Based on Carbon Nanotubes for Determination of Carbofuran in Human Serum by High Performance Liquid Chromatography.

Novel molecularly imprinted polymers (MIPs) based on multiwalled carbon nanotubes (MWNTs) were synthesized using carbofuran as template, methacrylic acid as functional monomer, and trimethylolpropane trimethacrylate as cross-liking agent, respectively. Characterization results showed that the carbofuran MIPs have been successfully grafted onto the surface of MWNTs as a thin layer with high stability. The results of adsorption dynamics indicated that the synthesized MWNT-MIPs displayed a biphase adsorption profile and good selective recognition to carbofuran with equilibrium adsorption of 106.2 mg/g. The MWNTs-MIPs synthesized were further applied as the adsorbent material of solid-phase extraction (SPE) for the pretreatment of carbofuran in human serum, analyzed using high performance liquid chromatograph (HPLC). The recoveries obtained ranged from 89.0 ± 4.8 to 93.6 ± 3.2, showing that the MWNTs-MIPs-SPE system developed have specific recognition toward carbofuran. Results above indicated that the proposed system filled with synthesized MWNTs-MIPs provided a fast and selective extraction of carbofuran in serum.

Development and validation of carbofuran and 3-hydroxycarbofuran analysis by high-pressure liquid chromatography with diode array detector (HPLC-DAD) for forensic Veterinary Medicine.

Agricultural pesticides used with the criminal intent to intoxicate domestic and wild animals are a serious concern in Veterinary Medicine. In order to identify the pesticide carbofuran and its metabolite 3- hydroxycarbofuran in animals suspected of exogenous intoxication a high pressure liquid chromatography with diode array detector (HPLC-DAD) method was developed and validated in stomach contents, liver, vitreous humor and blood. The method was evaluated using biological samples from seven different animal species. The following parameters of analytical validation were evaluated: linearity, precision, accuracy, selectivity, recovery and matrix effect. The method was linear at the range of 6.25-100µg/mL and the correlation coefficient (r2) values were >0.9811 for all matrices. The precision and accuracy of the method was determined by coefficient of variation (CV) and the relative standard deviation error (RSE), and both were less than 15%. Recovery ranged from 74.29 to 100.1% for carbofuran and from 64.72 to 100.61% for 3-hydroxycarbofuran. There were no significant interfering peaks or matrix effects. This method was suitable for detecting 25 positive cases for carbofuran amongst a total of 64 animal samples suspected of poisoning brought to the Toxicology Diagnostic Laboratory, School of Veterinary Medicine and Animal Sciences, University of Sao Paulo.

Poisoning by illegal rodenticides containing acetylcholinesterase inhibitors (chumbinho): a prospective case series.

OBJECTIVE: To describe a prospective case series of poisonings caused by ingestion of illegal rodenticides containing acetylcholinesterase inhibitors, mainly "chumbinho," followed-up by the Campinas PCC for a period of 1 year. CASE SERIES: Seventy-six cases were included, of which 53.9% were males. Age ranged from 2 to 74 years (median = 36 years). The main circumstances leading to poisoning were intentional (suicide attempts 92.1%; homicide attempts 5.3%), and 65.8% were admitted less than 2 hours after ingestion. Most of the patients (96.1%) showed cholinergic muscarinic manifestations, particularly salivation (86.8%), myosis (77.6%), sweating (50%), and bronchorrhea (35.5%). Atropine was used in 82.9% of patients (median = 2 days), intubation and mechanical ventilation in 46.1% (median = 3 days), and the median length of the hospital stay was 4 days. Plasma samples obtained upon admission in 59 cases revealed (LC-MS/MS): aldicarb (55), carbofuran (2), aldicarb and carbofuran (1), no active component (1). In most of the plasma and urine samples collected upon admission, the highest concentrations (ng/mL) obtained were for the active metabolite aldicarb sulphoxide (plasma, median = 831, IIQ = 99.2-2885; urine, median = 9800, IIQ = 2000-15000) than aldicarb (plasma, median = 237, IIQ = 35.7-851; urine, median = 584, IIQ = 166-1230), indicating rapid metabolism. The excretion of aldicarb and its metabolites was rapid since these compounds were rarely detected in plasma samples 48 hours after admission. Sequential cholinesterase analysis in 14 patients revealed almost complete reactivation in the first 48 hours post-admission, compatible for poisoning by carbamates. Based on the Poisoning Severity Score, the cases were classified as asymptomatic (5.3%), minor (11.8%), moderate (35.5%), severe (43.4%), and fatal (3.9%). CONCLUSIONS: Most poisonings involved aldicarb and resulted from suicide attempts; the poisonings were generally severe, with a mortality of 3.9%. Aldicarb was rapidly absorbed, metabolized, and excreted.

Investigation on possible ecotoxicological risk of carbofuran insecticides.

Carbofuran-containing insecticides are widely used agents in plant protection. Their use may pose considerable environmental risk for both the protected and non-protected predator and plantivorous birds. For defence of wild birds a model experiment was carried out on broiler chickens. In the study, eight animals were treated orally by gastric tube with a carbofuran-containing insecticide at a single dose of 2.5 mg/kg b.w. One animal served as untreated control specimen. Forage and drinking water were provided ad libitum. After the treatment, the possible clinical signs were observed carefully, blood samples were obtained from each bird and after exsanguinations liver, breast and leg muscle samples and stomach content were taken. The carbofuran concentration in blood, tissues and stomach content was determined by gas chromatographic method. Thirty minutes after poisoning, the average carbofuran concentration in breast muscle of chickens exceeded the maximum level of 0.1 mg/kg permitted in edible tissues, whereas ninety minutes after poisoning the concentration of one sample was still above the limit value. In the liver, leg-muscle and blood samples, the measured carbofuran concentration was lower than the permitted maximum value, except in the blood of two animals. The carbofuran concentration of the stomach content markedly exceeded the limit value. The sublethal concentration of the pesticides can reduce the capable of living of wild animals. Due to the sub toxic dose the poisoned birds can survive; however, the residue of insecticides can lead to secondary toxicosis of other animals.

A physiologically based pharmacokinetic/pharmacodynamic model for carbofuran in Sprague-Dawley rats using the exposure-related dose estimating model.

Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl-N-methylcarbamate), a broad spectrum N-methyl carbamate insecticide, and its metabolite, 3-hydroxycarbofuran, exert their toxicity by reversibly inhibiting acetylcholinesterase (AChE). To characterize AChE inhibition from carbofuran exposure, a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model was developed in the Exposure-Related Dose Estimating Model (ERDEM) platform for the Sprague-Dawley (SD) rat. Experimental estimates of physiological, biochemical, and physicochemical model parameters were obtained or based on data from the open literature. The PBPK/PD model structure included carbofuran metabolism in the liver to 16 known metabolites, enterohepatic circulation of glucuronic acid conjugates, and excretion in urine and feces. Bolus doses by ingestion of 50 microg/kg and 0.5 mg/kg carbofuran were simulated for the blood and brain AChE activity. The carbofuran ERDEM simulated a half-life of 5.2 h for urinary clearance, and the experimental AChE activity data were reproduced for the blood and brain. Thirty model parameters were found influential to the model outputs and were chosen for perturbation in Monte Carlo simulations to evaluate the impact of their variability on the model predictions. Results of the simulation runs indicated that the minimum AChE activity in the blood ranged from 29.3 to 79.0% (as 5th and 95th percentiles) of the control level with a mean of 55.9% (standard deviation = 15.1%) compared to an experimental value of 63%. The constructed PBPK/PD model for carbofuran in the SD rat provides a foundation for extrapolating to a human model that can be used for future risk assessment.

Determination of carbofuran, carbaryl and their main metabolites in plasma samples of agricultural populations using gas chromatography-tandem mass spectrometry.

A gas chromatography-tandem mass spectrometric (GC-MS/MS) method has been developed for the determination of carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate), carbaryl (1-naphthyl-N-methylcarbamate) and their main metabolites in human blood plasma. Optimization of the isolation of the compounds from plasma matrix included the precipitation, denaturation and digestion of plasma proteins. Derivatization was achieved by the use of trifluoroacetic acid anhydride and was optimized for temperature, time and volume of derivatization agent. In the proposed method, a mild precipitation technique was applied using beta-mercaptoethanol and ascorbic acid in combination with solid-phase extraction technique using Oasis HLB (Hydrophobic Lipophilic Balance) cartridges for further clean up of samples. Carbamate linkage was not hydrolyzed to its phenol product, but both carbamate phenol and ketones were transformed into trifluoroacetyl derivatives in order to become volatile compounds and were determined using tandem mass spectrometry. The linearity of the method was shown for nine concentrations in the range of 0.50-250 ng mL(-1) in fortified plasma aliquots. Limits of detection (LODs) for all compounds ranged from 0.015-0.151 ng mL(-1). Inter-day and intra-day assays (RSD) for all compounds, at three concentration levels of 2.5, 25 and 100 ng mL(-1) (n=3) in fortified plasma samples were less than 18%. Accuracy (%E (r)) was calculated at three concentration levels, 8, 80 and 160 ng mL(-1) (n=3), and ranged from -12.0 to 15.0%. Matrix effect was evaluated so mean recoveries were calculated for all compounds and ranged from 81-107%. Specificity for the use of this method to biological monitoring studies was achieved including four main metabolites of CF, 1-naphthol and 2-naphthol from the naphthalene metabolism pathways, and both the parent compound of carbofuran and carbaryl. The proposed method was applied to plasma samples of pesticide users.

[Detection of furadan in biological fluids].

Optimal conditions have been determined for furadan isolation from biological fluids by means of the mixture of solvents ethyl acetate-acetone in volume 1:1. Possible purification of the compound from coextractive substances of biomaterial on the column with silica gel L 100/160 mcm is demonstrated. IR-spectrophotometric methods and high performace liquid chromatography with a detector of photodiode matrix are proposed for identification and quantitation of furadan in blood and urine specimens.

Black-bellied whistling duck (Dendrocygna autumnalis) brain cholinesterase characterization and diagnosis of anticholinesterase pesticide exposure in wild populations from Mexico.

Rice is the main crop in the subbasin of the fluvial lagoon system of Palizada River (FLSPR) in the state of Campeche, Mexico. The pesticides used to control pests of this crop mainly are carbofuran, chlorpyrifos, and glyphosate. Black-bellied whistling duck (Dendrocygna autumnalis) is an ecologically and economically important species in the area. This duck is consumed by local inhabitants throughout the year, despite its potential exposure to pesticides. Due to its feeding habits, abundance, and nutritional value, D. autumnalis is a good indicator of environmental contamination and a potential route of human exposure to organophosphate and carbamate pesticides. In this study, the brain cholinesterase (ChE) in the frontal cerebral cortex of autochthonous ducks was characterized. In addition, the potential of the three locally used pesticides and mixtures to inhibit ChE activity was investigated and the exposure of the wild duck population during intensive pesticide applications in rice fields was evaluated. We found that acetylcholinesterase (AChE) seems to be the predominant ChE form in the biological fraction analyzed. Carbofuran was the most potent ChE inhibitor of D. autumnalis brain ChE activity from the three pesticides analyzed. Cholinesterase inhibition after exposure to pesticide mixtures predominantly was due to carbofuran. A decrease (p < 0.05) in AChE activity (>30%) was apparent in wild ducks compared to reference ducks, with recovery of ChE inhibition in wild ducks occurring months later when no pesticides were applied in the field. Dendrocygna autumnalis brain ChE is a suitable parameter for inclusion in biomonitoring programs for both environmental protection and human safety.

Dermal pharmacokinetics of the insecticide furathiocarb in rats.

The pharmacokinetics of furathiocarb were studied in vivo in male Sprague-Dawley rats following dermal treatment. HPLC and post-column derivatization were used for the analysis of furathiocarb and its metabolites (carbofuran, 3-hydroxycarbofuran and 3-ketocarbofuran). Carbofuran and 3-hydroxycarbofuran were detected in plasma and urine rather than furathiocarb. 3-Ketocarbofuran, another potential metabolite, was not observed in any sample. The concentration of carbofuran was higher than that of 3-hydroxycarbofuran in plasma, but the reverse was the case in urine. The corresponding area under the plasma concentration-time curve, Tmax, and Cmax values of carbofuran and 3-hydroxycarbofuran for 1500 mg kg-1 doses were 2.4-8.0 mg equiv hml-1, 12 h and 0.1-0.4 mg equiv ml-1, respectively. T1/2 was calculated only for 3-hydroxycarbofuran (28 h). Two metabolites were excreted in a dose-dependent manner without saturation.

Blood carbofuran concentrations in suicidal ingestion cases.

We describe four fatal cases due to ingestion of carbofuran, a carbamate insecticide. Carbofuran was detected in the gastric contents using thin layer chromatography (TLC) and gas chromatography/mass spectrophotometry (GC/MS), and quantified in the blood using a gas chromatograph equipped with nitrogen-phosphorus detector (NPD). Fatal concentrations of carbofuran in blood ranged from 0.32 to 11.6 microg/ml.

Poisoning from oral ingestion of carbofuran (Furadan 4F), a cholinesterase-inhibiting carbamate insecticide, and its effects on cholinesterase activity in various biological fluids.

A case is presented of a fatal ingestion of Furadan (carbofuran), a cholinesterase-inhibiting carbamate insecticide. A 26-year-old white male was found dead with a partially filled 1-gal (3.8-L) container of Furadan 4F insecticide-nematocide (44.9% carbofuran). The individual had ingested approximately 345 mL of the mixture. Analysis of cholinesterase activity in various biological fluids was performed spectrophotometrically using propionylthiocholine and 5,5'-dithiobis-2-nitrobenzoic acid [Sigma Diagnostics, cholinesterase procedure No. 422 (PTC)] which was measured at 405 nm and 30 degrees C in a Gilford Stasar III Spectrophotometer. The cholinesterase activities were as follows: plasma, 245 units (U)/L (93% inhibition/7% normal activity); serum, 208 U/L (95.3% inhibition/4.7% normal activity); whole blood, 297 U/L (92.8% inhibition/7.2% normal activity); erythrocytes, 58 U/L (99% inhibition/1% normal activity); vitreous humor, 7 U/L; and bile, 148 U/L. Carbofuran was detected in the blood and gastric contents by thin-layer chromatography. No alcohol or other drugs were detected in the blood, urine, or gastric contents. Ingestion of the carbofuran produced acute visceral congestion and pulmonary edema. Death was caused by anoxia due to respiratory paralysis produced by cholinesterase inhibition from Furadan (carbofuran) ingestion.

Relative inhibition of rat plasma and erythrocyte cholinesterases by pesticide combinations.

A selected combination of carbamate pesticides (carbofuran, oxamyl and propoxur were examined for cholinesterase (ChE) inhibition. The enzyme source was rat plasma and erythrocytes. Enzyme activity was determined colorimetrically using the Ellman technique adapted for erythrocyte ChE activity. Enzyme inhibition was obtained for both plasma and erythrocyte ChE, but inhibition by the pesticides was greater in the plasma. All 3 pesticides interacted additively in vitro.