Application of response surface methodology for optimization of trace amount of diazinon preconcentration in natural waters and biological samples by carbon mesoporous CMK-3.

Preconcentration of trace amounts of diazinon by carbon mesoporous CMK-3 in water and biological samples and measurement by high-performance liquid chromatography were investigated. CMK-3 was prepared using hexagonal SBA-15 as the template. The synthesized materials were characterized by X-Ray diffraction (XRD), Fourier transform infrared spectroscopy, Brunaur-Emmet-Teller, transmission electron microscopy and Boehm titration method. The preconcentration procedure was optimized using a multivariate optimization approach following a two-stage process. The effect of analytical parameters including the amount of the CMK-3 as an adsorbent, pH, type and volume of eluent and flow rate of eluent and sample were studied by a screening project, then the effective parameters were optimized by response surface methodology based on central composite design. The average extraction efficiency of diazinon under optimal conditions (CMK-3 dosage = 25 mg, sample flow rate = 2.5 mL min-1 , eluent flow rate = 1.25 mL min-1 , volume of methanol as an eluent =3.5 mL and initial pH = 6) was 97.11%, which agrees well with the predicted response value (97.93%). The linearity of the method was in the range of 0.5-100 µg L-1 with a correlation coefficient of 0.997. Enrichment factor, limit of detection and limit of quantification were 285.7, 0.09 and 0.23 µg L-1 , respectively. The relative standard deviation (RSD) under optimum conditions was 2.21% (n = 5). The proposed method was applied to determine diazinon in real water and biological samples. Recovery of diazinon from real samples was between 95.80 and 104.94% with an RSD of 0.19-4.65%. Thus, this method is suitable for the preconcentration and determination of diazinon in real water and biological samples.

[Determination of 15 kinds of pesticides in blood by gas chromatography-mass spectrometry].

OBJECTIVE: To develop the method of gas chromatography-mass spectrometry (GC-MS) for simultaneous determination of 15 herbicides in blood. METHODS: 2ml of blood in vitro were sampled, concentrated and extracted with dichloromethane, reconstant with methanol agents of Gulonic acid lactone solution, and detected by GC-MS. RESULTS: Experimental results show that diazinon, atrazine, prometryn, methyl parathion, butachlor, bifenthrin at 4-80 microg/L, phorate, malathion, 2,4-D butyl ester, chlordane, fenpropathrin at 10-200 microg/L, alpha-endosulfan, beta-endosulfan, cyhalothrin at 20-400 microg/L, dimethoate at 40-800 microg/L, with good linear response. The correlation coefficient (r2) were between 0.998-1.000, respectively. The recovery of all analysts averaged between 56%-128% in blood samples. The detection limits of all compounds between 0.05 and 1.00 microg/L. The lower limit of quantification between 0.20 and 3.001 microg/L. CONCLUSION: The methods is apply to detect the content of analysts in blood samples.

Organophosphate pesticide levels in blood and urine of women and newborns living in an agricultural community.

Organophosphate pesticides are widely used and recent studies suggest associations of in utero exposures with adverse birth outcomes and neurodevelopment. Few studies have characterized organophosphate pesticides in human plasma or established how these levels correlate to urinary measurements. We measured organophosphate pesticide metabolites in maternal urine and chlorpyrifos and diazinon in maternal and cord plasma of subjects living in an agricultural area to compare levels in two different biological matrices. We also determined paraoxonase 1 (PON1) genotypes (PON1(192) and PON1(-108)) and PON1 substrate-specific activities in mothers and their newborns to examine whether PON1 may affect organophosphate pesticide measurements in blood and urine. Chlorpyrifos levels in plasma ranged from 0-1,726 ng/mL and non-zero levels were measured in 70.5% and 87.5% of maternal and cord samples, respectively. Diazinon levels were lower (0-0.5 ng/mL); non-zero levels were found in 33.3% of maternal plasma and 47.3% of cord plasma. Significant associations between organophosphate pesticide levels in blood and metabolite levels in urine were limited to models adjusting for PON1 levels. Increased maternal PON1 levels were associated with decreased odds of chlorpyrifos and diazinon detection (odds ratio(OR): 0.56 and 0.75, respectively). Blood organophosphate pesticide levels of study participants were similar in mothers and newborns and slightly higher than those reported in other populations. However, compared to their mothers, newborns have much lower quantities of the detoxifying PON1 enzyme suggesting that infants may be especially vulnerable to organophosphate pesticide exposures.

Variation in plasma cholinesterase activity in the clay-colored robin (Turdus grayi) in relation to time of day, season, and diazinon exposure.

Cholinesterase (ChE) activity in birds is subject to interspecific and intraspecific species variations. Factors that influence enzyme activity have to be taken into account in order to obtain an accurate estimation of cholinesterase inhibition due to pesticide exposure in wild birds. This study evaluates variation of plasma cholinesterase activity in clay-colored robin (Turdus grayi) in relation to time of day, season, and exposure to diazinon. Other variables that can affect cholinesterase activity such as weight are also taken into account. The birds were marked, weighed and sexed using the cloacal technique. One dose of commercial diazinon mixed with papaya was fed to each bird at concentrations of 0.0, 0.5, 1.5 and 3.0 mg/kg ai. The results showed differences in ChE activity between seasons (t = -3.07, P < 0.05). Also, diurnal plasma cholinesterase variations were observed (20% in 2 h). The highest inhibition values were 73% for birds dosed with 1.5 mg/kg ai. Our study provides field and laboratory data on variation of ChE activity in a tropical bird species. Knowledge of the variation of ChE in the clay-colored robin will enable us to use this species as an indicator of exposure to ChE inhibiting pesticides in tropical agroecosystems.

A selective method for quantification of diazinon in human plasma by GC-MS.

Diazinon is a synthetic molecule known as an organophosphorus insecticide. It is used for gardens and for agriculture. This molecule represents a toxicological concern for humans. For this reason, the detection and the quantification of the diazinon in human samples are important in order to monitor an exposure and to diagnose intoxications. The aim of this work is to develop a selective method for the quantification of diazinon in human plasma by means of a gas chromatography (GC) with a mass spectroscopy (MS) detector. The method presented in this article includes a liquid-liquid extraction (LLE) using dichloromethane/propanol-2/heptane. The correlation coefficient for the calibration curve is 0.992. The limit of detection (LOD) and the limit of quantification (LOQ) are respectively 5 µg/L and 2 µg/L. The average recovery of the three different concentrations (15, 250 and 375 µg/L) varies from 22.8% to 31%. A test of stability at ambient temperature for 24 hours and at 4 °C for 48 hours was conducted. The relative error factor was found to be inferior to 15%. The results obtained show that the method developed gives satisfying validation parameters in human plasma. Therefore, the compound could be detected at very low concentration with a good linearity. This method can be used with other matrices, such as blood or urine, for a partial validation.

Liquid chromatography-tandem mass spectrometry method for the simultaneous quantitative determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood.

Simultaneous determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood by HPLC-tandem mass spectrometry was developed and validated. The pesticides were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Luna C(18) (30mmx2.0mm, 3microm) column, using a step-gradient at a flow rate of 0.4ml/min. The assay was linear from 0.5 to 100ng/ml (r(2)>0.992, n=24) for all pesticides. The inter- and intra-day accuracy and precision for the method was 96.6-106.1% and <10%, respectively. The lower limit of quantification was 0.5ng/ml. In conclusion, the method described displays analytical performance characteristics that are suitable for the quantification of these pesticides in cases of acute poisoning.

Pesticides contaminated dust exposure, risk diagnosis and exposure markers in occupational and residential settings of Lahore, Pakistan.

There are few studies documenting the dust loaded with pesticides as a potential non-dietary exposure source for occupational worker and populations living near agricultural farms and pesticides formulation plants. In present study we have evaluated the pesticide concentration in dust from potential sites and relevant health risk from dust ingestion. Furthermore, the effect of currently used pesticides was investigated on blood and urine parameters of subjects: farmer, factory worker, urban resident and rural resident and controlled subjects with presumably different levels of exposure. The urinary metabolites (TCPY and IMPY) were quantified as biomarkers of exposure to chlorpyrifos and diazinon in relation with biomarkers of effect including BuChE, LH, FSH, testosterone and oxidative stress. Results showed that chlorpyrifos and diazinon were present in higher concentration in dust and posed a high health risk to exposed subjects. The mean SOD value was high among the farmer (3048U/g Hb) followed by factory worker (1677.6U/g Hb). The urinary biomarkers - TCPY and IMPY- were found higher in exposed subjects as compared to control. Furthermore, testosterone was found in higher concentration in factory worker than control (12.63ng/ml vs 4.61ng/ml respectively). A decreased BuChE activity was noticed in occupational group and significant differences were observed in control verses exposed subjects. The PCA analysis evidenced the impact of pesticides on exposure biomarkers and male reproductive hormones. The study suggests that dust contaminated with pesticides engenders significant health risk particularly related to the nervous and endocrine system, not only for occupational workers exposed to direct ingestion but also for nearby residential community. Succinctly putting: Pesticides loaded dust in the city of Lahore, being a high priority concern for the government of Pakistan, demands to be addressed.

The assessment of occupational exposure to diazinon in Nicaraguan plantation workers using saliva biomonitoring.

A cross-sectional study with repeated sample collection in multiple days was conducted to assess diazinon exposures. Saliva and limited blood samples were collected from 10 banana plantation workers involved with diazinon application and their children aged 2-12 years living in Chinandega, Nicaragua. Diazinon concentration-time profiles in saliva varied between two plantations, which reflects the differences of work practices in each plantation. Salivary concentrations of diazinon measured in Plantation 1 applicators continued to increase 2 days after self-reported diazinon application, suggesting an ongoing exposure among these workers. However, salivary diazinon concentrations measured in Plantation 2 applicators were peaked 12 h prior to the first application, and then decreased 36 h post the first application. Diazinon concentrations in saliva were significantly correlated with the time-matched plasma samples collected from the same workers, which is in agreement with the previous published data from animal models. Children's exposure to diazinon through take-home pathway does not exist, as evident by the majority of nondetected saliva samples, and this finding was confirmed by the results from the urine samples. Severe dehydration was observed in many plantation workers and their children, resulting in the loss of some saliva samples, which no doubt have impaired the overall quality of the study results. Regardless, this article has demonstrated that saliva can be used to assess exposures to diazinon in pesticide applicators and children.

The tissue distribution of diazinon and the inhibition of blood cholinesterase activities in rats and mice receiving a single intraperitoneal dose of diazinon.

The tissue distribution of diazinon and the inhibition of cholinesterase (ChE) activities in plasma, erythrocyte and brain were investigated using male rats and mice which received a single intraperitoneal (i.p.) dose of diazinon (20 or 100 mg/kg body wt) in olive oil. The blood diazinon level was estimated to reach a maximum at 1-2 h after the i.p. administration. It was demonstrated that the diazinon residue levels are the highest in the kidney, when comparing the distribution of diazinon among liver, kidney and brain in the animals after dosing. It was indicated that the ChE inhibition by diazinon exposure is greater in the plasma than in the erythrocytes for male mice, while its inhibition is greater in the erythrocytes for male rats. Brain ChE activity was also inhibited markedly in the mice after dosing.

Chronic diazinon exposure: pathologies of spleen, thymus, blood cells, and lymph nodes are modulated by dietary protein or lipid in the mouse.

Little is known about the immunotoxicity of the organophosphate pesticide, diazinon. This study aims at detailing the pathologies in the thymus, spleen, blood cells, and lymph nodes (brachial, mesenteric, and hind quarter gluteal nodes) during chronic oral exposure (300 mg diazinonkg-1 food for 45 days), and explore the combined toxicity with excess dietary protein (40%) or lipid (20% corn oil). Animals were allowed to recover on normal food for 2 weeks. All experimental treatments caused organ pathologies, including necrotic degeneration of the trabeculae (spleen and thymus), hyperplasia of the cortex and medulla (thymus and lymph nodes), hyperplasia of white and red pulp (spleen), and sometimes haemorrhage (all tissues). Blood smears often showed crenated/hypochromic red cells and vacuolated white cells with abnormal nuclei. The severity of lesions during exposure was generally in the following order: lipid

Diazinon concentrations and blood cholinesterase activities in rats exposed to diazinon.

The tissue distribution of diazinon and the inhibition of blood cholinesterase (ChE) activity were investigated in male rats which received a single intraperitoneal (i.p.) dose of diazinon (100 mg/kg body wt) in olive oil. Diazinon concentration in the blood reached a maximum 1-2 h after dosing. Comparing the distribution of diazinon among liver, kidney and brain in treated rats, the diazinon residue was much greater in the kidney than in other organs, being 500 times that in the liver and 11 times that in the brain at 8 h after dosing. Erythrocyte and plasma ChE activities were inhibited rapidly, but ChE inhibition was greater in the erythrocytes than in plasma.

Biological monitoring of diazinon exposure using saliva in an animal model.

Alternative biological monitoring methods are currently being pursued to better quantify pesticide exposures. In this study, the feasibility of using saliva as a tool for measuring diazinon exposure was determined in an animal model. Male Spraque-Dawley rats were dosed with 1 or 10 mg/kg diazinon by bolus intravenous injection. Time-matched saliva and arterial blood samples were collected from 10 to 250 min post administration. Diazinon was distributed and eliminated rapidly in rats following intravenous (iv) bolus injection, according to a two-compartmental pharmacokinetic analysis. Salivary concentration of diazinon showed a strong correlation with plasma concentration of diazinon. The saliva/plasma (S/P) concentration ratio of diazinon was not affected by administered dose, sampling time, or salivary flow rate, suggesting that salivary excretion of diazinon in rats is fairly constant. Diazinon concentrations in saliva were consistently lower than those in arterial plasma. The mean S/P concentration ratios of diazinon were 0.16 and 0.13 for 1 and 10 mg/kg iv bolus doses, respectively. It is most likely that the incomplete transfer of diazinon from plasma to saliva is due to protein binding of diazinon in plasma. If the protein-unbound fraction of diazinon in plasma is used to calculate the S/P ratio, the S/P concentration ratio of diazinon is close to unity. The results from this study support the conclusion that diazinon salivary concentrations not only can be used to predict the plasma levels of diazinon in rats, but also reflect the unbound fraction of diazinon in plasma.

Rapid gas chromatographic assay of diazinon: application to the study of diazinon kinetics in rats.

A rapid method was developed for the determination of diazinon in plasma using gas chromatography with electron-capture detection (GC-ECD). Following a single extraction with hexane from 100 microliters of plasma, diazinon was quantitated on a 3% OV-17 column. The detection limit was 10 ng/ml and linearity was obtained in the range of 25 ng/ml-2500 ng/ml. The applicability of the assay to single-dose kinetics in rats is illustrated.

Diazinon toxicokinetics, tissue distribution and anticholinesterase activity in the rat.

The toxicokinetics, tissue distribution, and anticholinesterase (antiChE) activity of diazinon were investigated in the rat. Plasma concentrations most adequately fitted a two-compartment open model after i.v. administration of 10 mg/kg and a one-compartment model after oral administration of 80 mg/kg. Diazinon elimination half-life following i.v. and oral dosing was 4.70 and 2.86 h, respectively. The oral bioavailability was found to be low (35.5%). Hepatic extraction ratios after i.v. administration of 5 or 10 mg/kg were 54.8% and 47.7%, respectively, suggesting that low systemic oral bioavailability can be explained by a first-pass effect in the liver. Diazinon was found to be approximately 89% protein-bound in plasma within the concentration range 0.4-30 ppm. The highest concentration of diazinon after i.v. administration was found in the kidneys, when comparing to liver, kidney, brain. Both red blood cell (RBC) acetylcholinesterase (AChE) and plasma ChE activities were inhibited rapidly (44% and 17% at 10 min, and 36% and 13% min for i.v. and oral administration, respectively), but inhibition of RBC AChE was greater than that of plasma ChE.

Influence of cimetidine on the toxicity and toxicokinetics of diazinon in the rat.

1. The influence of cimetidine on diazinon toxicity and toxicokinetics was investigated in male Wistar rats. 2. The acute toxicity of diazinon, as well as brain acetylcholinesterase and carboxylesterase inhibition, were potentiated by pretreating rats with cimetidine (80 mg kg-1, i.p.) 1 and 24 h prior to diazinon application (50 mg kg-1, i.p.). 3. Comparison of toxicokinetic parameters between control and cimetidine-treated animals, showed a significant decrease in diazinon total body clearance and a marked increase in the area under the plasma concentration-time curve following cimetidine. 4. These results indicate that a major cause of the potentiation of diazinon may be related to the increase in the amount of diazinon in the systemic circulation as well as in the brain.

Diagnostic value of urinary dialkyl phosphate measurement in goats exposed to diazinon.

Recognition of exposure to diazinon, an organophosphate insecticide, was studied in goats. Urine and milk dialkyl phosphate concentrations (DETP; O,O-diethyl phosphorothionate) and blood cholinesterase activity (ChE) and diazinon concentrations were measured. Groups (n = 3 each) given (orally) diazinon at doses of 0.5 mg/kg for 7 days (small dose) or 5 mg/kg for 7 days (large dose) were compared with goats acutely exposed to single doses of 150 mg/kg (n = 1) or 700 mg/kg (n = 1). Clinical signs of intoxication occurred only in the goat given the 700 mg/kg dose. Urinary DETP concentrations were sensitive indicators of diazinon exposure and provided quantitative differences between small, large, and acute dosage exposures. Milk DETP concentrations were not detected. Cholinesterase measurement was useful only in the acute exposure studies. Whole blood diazinon concentrations were detected only in goats given the large dose for 7 days and acutely exposed. Measurement of urinary DETP was a sensitive aid for recognition of diazinon exposure.

Determination of diazinon, chlorpyrifos, and their metabolites in rat plasma and urine by high-performance liquid chromatography.

This study reports a simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of the insecticide diazinon (O,O-diethyl-O[2-isopropyl-6-methylpyridimidinyl] phosphorothioate), its metabolites diazoxon (O,O-diethyl-O-2-isopropyl-6-methylpyridimidinyl phosphate) and 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate), and TCP (3,5,6-trichloro-2-pyridinol) in rat plasma and urine samples. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction and HPLC with a reversed-phase C18 column and programmed UV detection ranging between 254 and 280 nm. The compounds are separated using a gradient of 1% to 80% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 mL/min in a period of 16 min. The limits of detection ranged between 50 and 150 ng/mL, and the limits of quantitation were 100 to 200 ng/mL. The average percentage recovery of five spiked plasma samples were 86.3 +/- 8.6, 77.4 +/- 7.0, 82.1 +/- 8.2, 81.8 +/- 8.7, 73.1 +/- 7.4, and 80.3 +/- 8.0 and from urine were 81.8 +/- 7.6, 76.6 +/- 7.1, 81.5 +/- 7.9, 81.8 +/- 7.1, 73.7 +/- 8.6, and 80.7 +/- 7.7 for diazinon, diazoxon, 2-isopropyl-6-methyl-4-pyrimidinol, chlorpyrifos, chlorpyrifos-oxon, and TCP, respectively. The relationship between the peak area and concentration was linear over a range of 200 to 2,000 ng/mL. This method was applied in order to analyze these chemicals and metabolites following dermal administration in rats.

Urinary determination of 2-isopropyl-4-methyl-6-hydroxypyrimidine in case of non fatal poisoning with diazinon.

We reported one non fatal case (42 month old boy) of intoxication with diazinon following accidental ingestion. Diazinon and three of its metabolites (2 common metabolites with other organophosphate pesticides: diethylphosphate and diethylthiophosphate; one specific metabolite: 2-isopropyl-4-methyl-6-hydroxypyrimidine) were determined in serum and in urine, respectively, using three liquid chromatography-tandem mass spectrometry methods. Diazinon was detected in serum while its metabolites were detected in urine. The concentrations of diazinon and its common metabolites were compared to concentrations previously described in literature in the same intoxication context and were discussed. The concentration of the specific metabolite was compared to concentrations highlighted in occupational exposure, because to the best of our knowledge, we reported here the first urinary concentration of this metabolite in an acute intoxication context.

Albumin binding as a potential biomarker of exposure to moderately low levels of organophosphorus pesticides.

We have evaluated the potential of plasma albumin to provide a sensitive biomarker of exposure to commonly used organophosphorus pesticides in order to complement the widely used measure of acetylcholinesterase (AChE) inhibition. Rat or human plasma albumin binding by tritiated-diisopropylfluorophosphate ((3)H-DFP) was quantified by retention of albumin on glass microfibre filters. Preincubation with unlabelled pesticide in vitro or dosing of F344 rats with pesticide in vivo resulted in a reduction in subsequent albumin radiolabelling with (3)H-DFP, the decrease in which was used to quantify pesticide binding. At pesticide exposures producing approximately 30% inhibition of AChE, rat plasma albumin binding in vitro by azamethiphos (oxon), chlorfenvinphos (oxon), chlorpyrifos-oxon, diazinon-oxon and malaoxon was reduced from controls by 9+/-1%, 67+/-2%, 56+/-2%, 54+/-2% and 8+/-1%, respectively. After 1 h of incubation with 19 microM (3)H-DFP alone, the level of binding to rat or human plasma albumins reached 0.011 or 0.039 moles of DFP per mole of albumin, respectively. This level of binding could be further increased by raising the concentration of (3)H-DFP, increasing the (3)H-DFP incubation time, or by substitution of commercial albumins for native albumin. Pesticide binding to albumin was presumed covalent since it survived 24 h dialysis. After dosing rats with pirimiphos-methyl (dimethoxy) or chlorfenvinphos (oxon) (diethoxy) pesticides, the resultant albumin binding were still significant 7 days after dosing. As in vitro, dosing of rats with malathion did not result in significant albumin binding in vivo. Our results suggest albumin may be a useful additional biomonitor for moderately low-level exposures to several widely used pesticides, and that this binding differs markedly between pesticides.

High performance liquid chromatographic determination of diazinon, permethrin, DEET (N, N-diethyl-m-toluamide), and their metabolites in rat plasma and urine.

A rapid method was developed for the analysis of the insecticide (A) diazinon (O,O-diethyl O-2-isopropyl-6-methylpyridimidinyl) phosphorothioate, its metabolites (B) diazoxon (O,O-diethyl O-2-isopropyl-6-methylpyridimidinyl) phosphate, and (C) 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide (D) permethrin [3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methylester], its metabolites (E) m-phenoxybenzyl alcohol, and (F) m-phenoxybenzoic acid, the insect repellent (G) DEET (N,N-diethyl-m-toluamide), and its metabolites (H) m-toluamide and (I) m-toluic acid in rat plasma and urine. The method is based on using C18 Sep-Pak cartridges (Waters Corporation, Milford, Mass., U.S.A.) for solid phase extraction and high performance liquid chromatography with a reversed phase C18 column, and absorbance detection at 230 nm for compounds A, B, and C, and at 210 nm for compounds D-I. The compounds were separated using a gradient from 1% to 99% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.7 mL/min in a period of 17 min. The limits of detection were ranged between 20 and 100 ng/mL, while limits of quantification were 80-200 ng/mL. The relationship between peak areas and concentration was linear over a range of 100-1000 ng/mL. This method was applied to determine the above insecticides and their metabolites following dermal administration in rats.