Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition

Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphorothioyloxy)benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-linked immunosorbent assay (ciELISA) using a heterologous coating antigen, 4-(3-(diethoxyphosphorothioyloxy)phenylamino)-4-oxobutanoic acid. The 50% inhibition value (IC₅₀) was 348 ng mL⁻¹ for parathion, 13 ng mL⁻¹ for coumaphos, 22 ng mL⁻¹ for quinalphos, 35 ng mL⁻¹ for triazophos, 751 ng mL⁻¹ for phorate, 850 ng mL⁻¹ for dichlofenthion, and 1301 ng mL⁻¹ for phoxim. The limit of detection (LOD) met the ideal detection criteria of all the seven OP residues. A quantitative structure-activity relationship (QSAR) model was constructed to study the mechanism of antibody recognition using multiple linear regression analysis. The results indicated that the frontier-orbital energies (energy of the highest occupied molecular orbital, E_HOMO, and energy of the lowest unoccupied molecular orbital, E_LUMO) and hydrophobicity (log of the octanol/water partition coefficient, log P) were mainly responsible for the antibody recognition. The linear equation was log(IC₅₀) = −63.274E_HOMO + 15.985E_LUMO + 0.556 log P − 25.015, with a determination coefficient (r²) of 0.908.     

Development of an extraction method for the determination of prostaglandins in biological tissue samples using liquid chromatography–tandem mass spectrometry: Application to gonads of Atlantic cod (Gadus morhua)

A simple extraction method for the analysis of PGE₂ and PGF_2α in gonad samples from Atlantic cod and further quantification by using liquid chromatography–tandem mass spectrometry is proposed. The evaluation of the best solvent extraction conditions and the analytical performance parameters are reported. The method was highly selective for both prostaglandins and the calibration curves, based on the internal standard method, were linear between 5 and 1000 ng mL⁻¹ for PGE₂ and PGF_2α, with limits of detection of 1 ng mL⁻¹ and 1.5 ng mL⁻¹ and recovery values of 99.999 ± 0.002 and 99.967 ± 0.023 respectively. The homogenization of samples using liquid nitrogen combined with the developed extraction protocol can be implemented in different types of biological tissues.     

HPLC-UV and HPLC-MSn multiresidue determination of amidosulfuron, azimsulfuron, nicosulfuron, rimsulfuron, thifensulfuron methyl, tribenuron methyl and azoxystrobin in surface waters

The paper presents a new HPLC method, with UV and MSⁿ detection, for the determination of seven pesticides, including the sulfonylurea herbicides amidosulfuron, azimsulfuron, nicosulfuron, rimsulfuron, thifensulfuron methyl, tribenuron methyl, and the fungicide azoxystrobin characterised by a methoxyacrilate structure. The methodology consists of a preconcentration/SPE (solid phase extraction) step and HPLC-UV (240 nm detection wavelength)-MSⁿ analysis. Under the optimised conditions and after a 1000/1 preconcentration factor, the limits of detection were lower than 14.5 ng L⁻¹ for UV detection and lower than 8.1 ng L⁻¹ for MS detection. The limits of quantification were lower than 48.3 ng L⁻¹ in UV detection and than 26.9 ng L⁻¹ in MSⁿ detection. The analysis of two samples, spiked with a mixture of the pesticides at threshold level concentrations, gave more than 60% recovery.     

Ultrasound-assisted surfactant-enhanced emulsification microextraction for the determination of carbamate pesticides in water samples by high performance liquid chromatography

A novel ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME) coupled with high performance liquid chromatography-diode array detection has been developed for the extraction and determination of six carbamate pesticides (metolcarb, carbofuran, carbaryl, pirimicarb, isoprocarb and diethofencarb) in water samples. In the UASEME technique, Tween 20 was used as emulsifier, and chlorobenzene and chloroform were used as dual extraction solvent without using any organic dispersive solvent that is normally required in the previously described common dispersive liquid–liquid microextraction method. Parameters that affect the extraction efficiency, such as the kind and volume of the extraction solvent, the type and concentration of the surfactant, ultrasound emulsification time and salt addition, were investigated and optimized for the method. Under the optimum conditions, the enrichment factors were in the range between 170 and 246. The limits of detection of the method were 0.1–0.3 ng mL⁻¹ and the limits of quantification were between 0.3 and 0.9 ng mL⁻¹, depending on the compounds. The linearity of the method was obtained in the range of 0.3–200 ng mL⁻¹ for metolcarb, carbaryl, pirimicarb, and diethofencarb, 0.6–200 ng mL⁻¹ for carbofuran, and 0.9–200 ng mL⁻¹ for isoprocarb, with the correlation coefficients (r) ranging from 0.9982 to 0.9998. The relative standard deviations varied from 3.2 to 4.8% (n = 5). The recoveries of the method for the six carbamates from water samples at spiking levels of 1.0, 10.0, 50.0 and 100.0 ng mL⁻¹ were ranged from 81.0 to 97.5%. The proposed UASEME technique has demonstrated to be simple, practical and environmentally friendly for the determination of carbamates residues in river, reservoir and well water samples.     

Ligandless-dispersive liquid-liquid microextraction of trace amount of copper ions

In the present work, a new ligandless-dispersive liquid-liquid microextraction (LL-DLLME) method has been developed for preconcentration trace amounts of copper as a prior step to its determination by flame atomic absorption spectrometry. In the proposed approach 1,2-dicholorobenzene and ethanol were used as extraction and dispersive solvents, respectively. Some factors influencing on the extraction efficiency of copper and its subsequent determination were studied and optimized, such as the extraction and dispersive solvent type and volume, pH of sample solution, extraction time and salting out effect. Under the optimal conditions, the calibration curve was linear in the range of 1.0 ng mL⁻¹-0.6 μg mL⁻¹ of copper with R² = 0.9985. Detection limit was 0.5 ng mL⁻¹ in original solution (3S_b/m) and the relative standard deviation for seven replicate determination of 0.2 μg mL⁻¹ copper was ±1.4%. The proposed method has been applied for determination of copper in standard and water samples with satisfactory results.     

Adsorptive stripping square wave voltammetry (Ad-SSWV) accomplished with second-order multivariate calibration determination of fenitrothion and its metabolites in river water samples

A method, using stripping square wave voltammetry (Ad-SSWV), for the simultaneous determination of fenitrothion (FEN) and its metabolites: fenitrooxon (OXON) and 3-methyl-4-nitrophenol (3-MET) in environmental samples is reported. All three compounds produce, at mercury electrode (HMDE), an electrochemical signal due to an adsorptive-reductive process. The electrochemical approach shows a very high overlap degree for FEN and OXON voltammograms, however the adsorption kinetic profile could be used as an additional differential variable between both analytes. Second-order multivariate calibration has been tested to solve the mixture of the three compounds. The second-order assayed methods were parallel factor analysis (PARAFAC), unfolded partial least squares (U-PLS), multidimensional partial least squares (N-PLS) and the latest ones were used in combination with the residual bilinearization procedure RBL. U-PLS/RBL model was stated as the best second-order algorithm for the simultaneous determination of these three compounds up to 50 ng mL⁻¹ for each analyte. The detection limits and recovery values were 1.6 ng mL⁻¹ and 92 ± 7% for FEN; 3.7 ng mL⁻¹ and 101 ± 9% for OXON and 0.6 ng mL⁻¹ and 97 ± 8% for 3-MET.     

Ligandless dispersive liquid-liquid microextraction for the separation of trace amounts of silver ions in water samples and flame atomic absorption spectrometry determination

In this article, a new ligandless dispersive liquid-liquid microextraction method has been developed for preconcentration of trace quantities of silver as a prior step to its determination by flame atomic absorption spectrometry. In the proposed approach, carbon tetrachloride and ethanol were used as extraction and dispersive solvents. Several factors that may be affected on the extraction process, like, extraction solvent, disperser solvent, the volume of extraction and disperser solvent, pH of the aqueous solution and extraction time were optimized. Under the optimal conditions, the calibration curve was linear in the range of 5.0 ng mL⁻¹ to 2.0 μg mL⁻¹ of silver with R² = 0.9995 (n = 9) and detection limit based on three times the standard deviation of the blank (3S_b) was 1.2 ng mL⁻¹ in original solution. The relative standard deviation for eight replicate determination of 0.5 μg mL⁻¹ silver was ±1.5%. The high efficiency of dispersive liquid-liquid microextraction to carry out the determination of silver in complex matrices was demonstrated. The proposed method has been applied for determination of trace amount of silver in standard and water samples with satisfactory results.     

Application of dispersive liquid-liquid microextraction for the analysis of triazophos and carbaryl pesticides in water and fruit juice samples

In this work, a simple, rapid and sensitive sample pretreatment technique, dispersive liquid-liquid microextraction (DLLME) coupled with high performance liquid chromatography-fluorescence detection (HPLC-FLD), has been developed to determine carbamate (carbaryl) and organophosphorus (triazophos) pesticide residues in water and fruit juice samples. Parameters, affecting the DLLME performance such as the kind and volume of extraction and dispersive solvents, extraction time and salt concentration, were studied and optimized. Under the optimum extraction conditions (extraction solvent: tetrachloroethane, 15.0 μL; dispersive solvent: acetonitrile, 1.0 mL; no addition of salt and extraction time below 5 s), the performance of the proposed method was evaluated. The enrichment factors for the carbaryl and triazophos were 87.3 and 275.6, respectively. The linearity was obtained in the concentration range of 0.1-1000 ng mL⁻¹ with correlation coefficients from 0.9991 to 0.9999. The limits of detection (LODs), based on signal-to-noise ratio (S/N) of 3, ranged from 12.3 to 16.0 pg mL⁻¹. The relative standard deviations (RSDs, for 10 ng mL⁻¹ of carbaryl and 20 ng mL⁻¹ of triazophos) varied from 1.38% to 2.74% (n = 6). The environmental water (at the fortified level of 1.0 ng mL⁻¹) and fruit juice samples (at the fortified level of 1.0 and 5.0 ng mL⁻¹) were successfully analyzed by the proposed method, and the relative recoveries of them were in the range of 80.4-114.2%, 89.8-117.9% and 86.3-105.3%, respectively.     

Multiresidue method for the determination of 13 pesticides in three environmental matrices: water, sediments and fish muscle

Pesticides residues in aquatic ecosystems are an environmental concern which requires efficient analytical methods. In this study, we proposed a generic method for the quantification of 13 pesticides (azoxystrobin, clomazone, diflufenican, dimethachlor, carbendazim, iprodion, isoproturon, mesosulfuron-methyl, metazachlor, napropamid, quizalofop and thifensulfuron-methyl) in three environmental matrices. Pesticides from water were extracted using a solid phase extraction system and a single solid-liquid extraction method was optimized for sediment and fish muscle, followed by a unique analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Limits of quantification were below 5 ng L⁻¹ for water (except for fluroxypyr and iprodion) and ranged between 0.1 ng g⁻¹ and 57.7 ng g⁻¹ for sediments and regarding fish, were below 1 ng g⁻¹ for 8 molecules and were determined between 5 and 49 ng g⁻¹ for the 5 other compounds. This method was finally used as a new routine practice for environmental research.     

Determination of selenium in water and soil by hydride generation atomic absorption spectrometry using solid reagents

A hydride generation method for the determination of traces of selenium at ng mL⁻¹ concentration ranges has been introduced using a solid mixture of tartaric acid and sodium tetrahydroborate. Atomic absorption spectrometry (AAS) has been used as the detection system. Several parameters such as the ratio of tartaric acid to sodium tetrahydroborate, type and amount of acid, and the reaction temperature were optimized by using 640 ng mL⁻¹ (16 ng per 25 μL) of Se(IV) standard solution. The calibration curve was linear from 20 to 1200 ng mL⁻¹ (0.5-30 ng Se(IV) per 25 μL). The relative standard deviation (%R.S.D.) of the determination was 1.93% and the detection limit was 10.6 ng mL⁻¹ (265 pg per 25 μL) of Se(IV). The reliability of the method was checked using different types of environmental samples, such as several types of water, a sample of soil and also in a kind of calcium phosphate sample by standard addition method. For conversion of Se(VI) present in real samples to Se(IV), l-cysteine was added to NaBH₄ and tartaric acid mixture. The results showed good agreement between this method and other hydride generation techniques.     

Dispersive liquid-liquid microextraction combined with high performance liquid chromatography-fluorescence detection for the determination of carbendazim and thiabendazole in environmental samples

A rapid and sensitive method for the determination of carbendazim (methyl benzimidazole-2-ylcarbamate, MBC) and thiabendazole (TBZ) in water and soil samples was developed by using dispersive liquid-liquid microextraction (DLLME) coupled with high performance liquid chromatography with fluorescence detection. The water samples were directly used for the DLLME extraction. For soil samples, the target analytes were first extracted by 0.1 mol L⁻¹ HCl. Then, the pH of the extract was adjusted to 7.0 with 2 mol L⁻¹ NaOH before the DLLME extraction. In the DLLME extraction method, chloroform (CHCl₃) was used as extraction solvent and tetrahydrofuran (THF) as dispersive solvent. Under the optimum conditions, the enrichment factors for MBC and TBZ were ranged between 149 and 210, and the extraction recoveries were between 50.8 and 70.9%, respectively. The linearity of the method was obtained in the range of 5-800 ng mL⁻¹ for water sample analysis, and 10-1000 ng g⁻¹ for soil samples, respectively. The correlation coefficients (r) ranged from 0.9987 to 0.9997. The limits of detection were 0.5-1.0 ng mL⁻¹ for water samples, and 1.0-1.6 ng g⁻¹ for soil samples. The relative standard deviations (RSDs) varied from 3.5 to 6.8% (n = 5). The recoveries of the method for MBC and TBZ from water samples at spiking levels of 5 and 20 ng mL⁻¹ were 84.0-94.0% and 86.0-92.5%, respectively. The recoveries for soil samples at spiking levels of 10 and 100 ng g⁻¹ varied between 82.0 and 93.4%.     

Dispersive liquid-liquid microextraction versus single-drop microextraction for the determination of several endocrine-disrupting phenols from seawaters

Two liquid-phase microextraction procedures: single-drop microextraction (SDME) and dispersive liquid-liquid microextraction (DLLME), have been developed for the determination of several endocrine-disrupting phenols (EDPs) in seawaters, in combination with high-performance liquid chromatography (HPLC) with UV detection. The EDPs studied were bisphenol-A, 4-cumylphenol, 4-tertbutylphenol, 4-octylphenol and 4-n-nonylphenol. The optimized SDME method used 2.5 μL of decanol suspended at the tip of a micro-syringe immersed in 5 mL of seawater sample, and 60 min for the extraction time. The performance of the SDME is characterized for average relative recoveries of 102 ± 11%, precision values (RSD) < 9.4% (spiked level of 50 ng mL⁻¹), and detection limits between 4 and 9 ng mL⁻¹. The optimized DLLME method used 150 μL of a mixture acetonitrile:decanol (ratio 15.7, v/v), which is quickly added to 5 mL of seawater sample, then subjected to vortex during 4 min and centrifuged at 2000 rpm for another 5 min. The performance of the DLLME is characterized for average relative recoveries of 98.7 ± 3.7%, precision values (RSD) < 7.2% (spiked level of 20 ng mL⁻¹), and detection limits between 0.2 and 1.6 ng mL⁻¹. The efficiencies of both methods have also been compared with spiked real seawater samples. The DLLME method has shown to be a more efficient approach for the determination of EDPs in seawater matrices, presenting enrichment factors ranging from 123 to 275, average relative recoveries of 110 ± 11%, and precision values (RSD) < 14%, when using a real seawaters (spiked level of 3.5 ng mL⁻¹).     

An enzymeless organophosphate pesticide sensor using Au nanoparticle-decorated graphene hybrid nanosheet as solid-phase extraction

A sensitive enzymeless organophosphate pesticides (OPs) sensor is fabricated by using Au nanoparticles (AuNPs) decorated graphene nanosheets (GNs) modified glassy carbon electrode as solid phase extraction (SPE). Such a nanostructured composite film, combining the advantages of AuNPs with two dimensional GNs, dramatically facilitates the enrichment of nitroaromatic OPs onto the surface and realizes their stripping voltammetric detection of OPs by using methyl parathion (MP) as a model. The stripping voltammetric performances of captured MP were evaluated by cyclic voltammetric and square-wave voltammetric analysis. The combination of the nanoassembly of AuNPs-GNs, SPE, and stripping voltammetry provides a fast, simple, and sensitive electrochemical method for detecting nitroaromatic OPs. The stripping analysis is highly linear over the MP concentration ranges of 0.001-0.1and 0.2-1.0 μg mL⁻¹ with a detection limit of 0.6 ng mL⁻¹. This designed enzymeless sensor exhibits good reproducibility and acceptable stability.     

Dispersive liquid-liquid microextraction followed by reversed phase-high performance liquid chromatography for the determination of polybrominated diphenyl ethers at trace levels in landfill leachate and environmental water samples

A simple, rapid and efficient method, dispersive liquid-liquid microextraction (DLLME), has been developed for the extraction and preconcentration of polybrominated diphenyl ethers (PBDEs) in water samples. The factors influencing microextraction efficiencies, such as the kind and volume of extraction and dispersive solvent, the extraction time and the salt effect, were optimized. Under the optimum conditions (sample volume: 5 mL; extraction solvent: tetrachloroethane, 20.0 μL; dispersive solvent: acetonitrile, 1.00 mL; extraction time: below 5 s and without salt addition), the enrichment factors and extraction recoveries were high and ranged from 268 to 305 and 87.0 to 119.1%, respectively. Linearity was observed in the range 0.05-50 ng mL⁻¹ for BDE-28 and BDE-99, and 0.1-100 ng mL⁻¹ for BDE-47 and BDE-209, respectively. Coefficients of correlation (r²) ranged from 0.9995 to 0.9999. The repeatability study was carried out by extracting the spiked water samples at concentration levels of 50 ng mL⁻¹ for BDE-28 and BDE-99, and 100 ng mL⁻¹ for BDE-47 and BDE-209, respectively. The relative standard deviations (R.S.D.s) varied between 3.8 and 6.3% (n = 5). The limits of detection (LODs), based on signal-to-noise ratio (S/N) of 3, ranged from 12.4 to 55.6 pg mL⁻¹ (the wavelength of detector at 226 nm). The relative recoveries of PBDEs from tap, lake water and landfill leachate samples at spiking levels of 5, 10 and 50 ng mL⁻¹ were in the range of 89.7-107.6%, 114.3-119.1% and 87.0-90.9%, respectively. As a result, this method can be successfully applied for the determination of PBDEs in landfill leachate and environmental water samples.     

Headspace solid-phase microextraction using a dodecylsulfate-doped polypyrrole film coupled to ion mobility spectrometry for the simultaneous determination of atrazine and ametryn in soil and water samples

A simple and rapid headspace solid-phase microextraction (HS-SPME) based method is presented for the simultaneous determination of atrazine and ametryn in soil and water samples by ion mobility spectrometry (IMS). A dodecylsulfate-doped polypyrrole (PPy-DS), synthesized by electrochemical method, was applied as a laboratory-made fiber for SPME. The HS-SPME system was designed with a cooling device on the upper part of the sample vial and a circulating water bath for adjusting the sample temperature. The extraction properties of the fiber to spiked soil and water samples with atrazine and ametryn were examined, using a HS-SPME device and thermal desorption in injection port of IMS. Parameters affecting the extraction efficiency such as the volume of water added to the soil, pH effect, extraction time, extraction temperature, salt effect, desorption time, and desorption temperature were investigated. The HS-SPME-IMS method with PPy-DS fiber, provided good repeatability (RSDs < 10 %), simplicity, good sensitivity and short analysis times for spiked soil (200 ng g⁻¹) and water samples (100 and 200 ng mL⁻¹). The calibration graphs were linear in the range of 200-4000 ng g⁻¹ and 50-2800 ng mL⁻¹ for soil and water respectively (R² > 0.99). Detection limits for atrazine and ametryn were 37 ng g⁻¹ (soil) and 23 ng g⁻¹ (soil) and 15 ng mL⁻¹ (water) and 10 ng mL⁻¹ (water), respectively. To evaluate the accuracy of the proposed method, atrazine and ametryn in the three kinds of soils and two well water samples were determined. Finally, comparing the HS-SPME results for extraction and determination of selected triazines using PPy-DS fiber with the other methods in literature shows that the proposed method has comparable detection limits and RSDs and good linear ranges.     

Low-density extraction solvent-based solvent terminated dispersive liquid–liquid microextraction combined with gas chromatography-tandem mass spectrometry for the determination of carbamate pesticides in water samples

A simple and fast method of low-density extraction solvent-based solvent terminated dispersive liquid–liquid microextraction (ST-DLLME) was developed for the highly sensitive determination of carbamate pesticides in the water samples by gas chromatography-tandem mass spectrometry (GC-MSMS). After dispersing, the obtained emulsion cleared into two phases quickly when an aliquot of acetonitrile was introduced as a chemical demulsifier into the aqueous bulk. Therefore, the developed procedure does not need centrifugation to achieve phase separation. It was convenient for the usage of low-density extraction solvents in DLLME. Under the optimized conditions, the limits of detection for all target carbamate pesticides were in range of 0.001–0.50 ng mL⁻¹ and the precisions were in the range of 2.3–6.8% (RSDs, 2 ng mL⁻¹, n = 5). The proposed method has been successfully applied to the analysis of real water samples and good spiked recoveries over the range of 94.5–104% were obtained.     

Leachability and analytical speciation of antimony in coal fly ash

A new procedure was described with multiwalled carbon nanotubes as solid phase extraction packing material for the trace analysis of nicosulfuron, thifensulfuron and metsulfuron-methyl in water samples. The possible parameters influencing the enrichment were optimized and the optimal conditions were as followed: eluent, sample pH, flow rate and sample volume were acetonitrile containing 1% acetic acid, pH 3, 8 mL min⁻¹ and 500 mL, respectively. Under the optimal chromatographic separation and SPE conditions, the linear range, detection limit (S/N = 3) and precision (R.S.D., n = 6) were 0.04-40 ng mL⁻¹, 6.8 ng L⁻¹ and 2.5% for nicosulfuron, 0.04-40 ng mL⁻¹, 11.2 ng L⁻¹ and 5.4% for thifensulfuron, 0.02-20 ng mL⁻¹, 5.9 ng L⁻¹, 2.1% for metsulfuron-methyl, respectively. The established method was well employed to determine nicosulfuron, thifensulfuron and metsulfuron-methyl in tap water, seawater, reservoir water and well water samples, and satisfactory results were obtained, the spiked recoveries in the range of 87.2-100.7%, 96.5-105.6% and 83.7-111.1% for them each, respectively.     

Determination of atrazine and simazine in water samples by high-performance liquid chromatography after preconcentration with heat-treated diatomaceous earth

A sensitive and selective column adsorption method is proposed for the preconcentration and determination of atrazine and simazine. Atrazine and simazine were preconcentrated on heat-treated diatomaceous earth as an adsorbent and then determined by high-performance liquid chromatography (HPLC). Several parameters on the recoveries of the analytes were investigated. The experimental results showed that it was possible to obtain quantitative analysis when the solution pH was 2 using 100 mL of validation solution containing 1.5 μg of triazines and 5 mL of ethanol as an eluent. Recoveries of atrazine and simazine were 95.7 ± 4.2% and 75.0 ± 1.9% with a relative standard deviation for seven determinations of 4.7% and 2.7% under optimum conditions. The maximum preconcentration factor was 100 for triazines when 500 mL of sample solution volume was used. The linear ranges of calibration curves for atrazine and simazine were 1-150 ng mL⁻¹ and 1-300 ng mL⁻¹, respectively, with correlation coefficients of 0.999 and the detection limits (3Signal-to-Noise) were 0.24 ng mL⁻¹ and 0.21 ng mL⁻¹ for atrazine and simazine. The capacity of the adsorbent was also examined and found to be 0.8 mg g⁻¹ and 1.3 mg g⁻¹ for atrazine and simazine, respectively. The proposed method was successfully applied to the determination of triazines in river water and tap water samples with high precision and accuracy.     

Enzyme-linked immunosorbent assays for the insecticide fenitrothion. Influence of hapten conformation and sample matrix on assay performance

This study aimed at developing competitive enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus (OP) insecticide fenitrothion using a monoclonal antibody. The hapten used to obtain the antibody had an ideal structural feature that allowed minimal functional group sacrifice. By using the antibody and a coating antigen, a competitive indirect ELISA was developed, which showed an IC₅₀ of 14 ng mL⁻¹ with a detection limit of 3.0 ng mL⁻¹. A competitive direct ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 17 ng mL⁻¹ with a detection limit of 1.6 ng mL⁻¹. The antibodies in both assays showed negligible cross-reactivity with the metabolites of fenitrothion and other OP pesticides except with the insecticides parathion-methyl and parathion-ethyl. Recoveries of fenitrothion from fortified rice and lettuce samples were determined and the bias in the recovery values was rationalized by using the standard curves obtained in the matrix extract.     

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL⁻¹ for urine and 20 ng mL⁻¹ for plasma. The limit of detection and limit of quantification were 0.13 ng mL⁻¹ and 2.5 ng mL⁻¹, respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL⁻¹, but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng μmol⁻¹ creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.