Determinition Method And Dissipation Of New Hebercide H9201 Under Crop Planting Conditions

Agrochemicals play important roles in modern agriculture; however, concerns about environmental impact of agrochemical and risk to human health have increased. Due to the intellectual property rights, research aiming at the development of enviromental friendly and safely agri-chemicals is increasingly important. H9201 is a new agent for the weed control, which was invented by the State Key Lab of Element-organic Chemistry (Nankai University), and performed broad-spectrum weed control activity under water field and dry field conditions. Hereby, our study focused on the analytical method development and enviromental fate analysis and dissipation pathway profiling of this new agent in order to provide basic information for the evaluation of risk to human health, which will help to determine the maxiam residue limit value (MRL). Our research conducted includes method development (polycolonal antibody development for the ELISA method establishment, GC, GC/MS, and LC/ESI/ITMS analytical method development), and residual dynamics of H9201 in environment after field application. The main results are listed as follows.1. Indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) development.An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for H9201 determination was developed based on polyclonal antibody, and was employed in the sample analysis.Chemical O-methyl-O-(2,4-dimethyl-6-nitrophenoxy)-N-phosphoramide (hapten) was synthesized by thiophosphoryl chloride and 2,4-dimethyl-6-nitrophenol, then succinic anhydride was used to modify amino group with Dimethylamino pyridine (DMAP) catalysising, thereafter conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) by active ester method (AEM). Final conjugation identified by UV screen and SDS-PAGE, the conjugate ratio of Hapten-BSA is:42.7:1; Hapten-OVA is:18.2:1, respectively. Polyclonal antibodies raised against Hapten-BSA were screened and selected for the IC-ELISA. One set of serum from the rabbit H1600 showed an I50 value of 5.8551μg/mL with a detection limit of 0.2291μg/mL at the concentration of H9201 ranged from 0.6508-38.1422μg/mL, and the serum raised from the rabbit H6000 had an I50 value of 3.2765μg/mL with a detection limit of 0.2007μg/mL at the range of H9201 concentration from 0.3204 to 18.0652μg/mL. A sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of new herbicide H-9201 based on the polyclonal antibody H6600 generated in rabbits by immunizing with synthesized hapten. For standard,50% reduction of the maximum ELISA signal (IC50) was 1.331μg/mL, whereas the detection limit was 0.017μg/mL in the competitive standard curve ranged from 0.05μg/mL to 20.0μg/mL, which combination of the concentrations of coating solution and primary antibody was optimized to 1/512 and 1/18 000, respectively. No significant cross-reactivity for related compounds was observed, such as DMNT, p-nitrophenol, Methamidophos, Phorate, Chlorpyrifos, Parathion, the antibody showed negligible cross-reactivity with S-Carboxylethyl-O,O-Dimethyl-phosphorodithioate, dimethoate, malathion, but high cross-reactivity with analogs of hapten. Recovery of H-9201 in environmental water,soil and carrot based on the developed IC-ELISA method with sample previous treatment step was 78.4±2.4%～95.2±3.2%,72.1±4.8%～85.4±5.1%,74.4±4.3%～91.3±4.2%,respectively.2. Method development of GC, GC/MS, LC/MS for H9201 residue determination.Method Development based on Gas Chromatergraphy with frame photometric detector (FPD). DB-17 (1μm film thickness,30 m length,0.53 mm ID, J&W Scientific, USA) column was used for the analyte analysis, GC work conditions as follows:the inlet temperature was 250℃and the injection was carried out in splitless modle using a 1μL injection volume. For FPD, the detector temperature was 200℃, the initial oven temperature was 150℃, and was maintained for 2 min, the temperature was then increased 8℃/min to 250℃and held for 12 min.The recoveries ranged from 90.8～98.4% in soil matrix,89.2～101.8% in carrot matrix,97.43～100.91% in water,96.37～115.39% in natural pond water, respectively.The limit of detection of method is 0.01 mg/kg in soil,0.02 mg/kg in carrot, and 0.008 mg/kg in tap water,0.02 mg/kg in natural pond water, respectively.HP-5MS (0.25μm film thickness,30 m length,0.32 mm ID, J&W Scientific, USA) connected with MS for GC-MS method development. The gas chromatograph settings were the same as described above. The ionization potential of mass selective detector was 70 eV and the scan range was 20-450 m/z, the ion-source temperature 230℃, and the GC-MS interface temperature 280℃. Selected ion monitoring was performed with target ion 318.3, one quantifying ion 272(100), and two qualifying ions 230 (32),152(22) for analysis, Helium was used as the carrier gas at a flow rate of 1.0 mL·min-1. All gases were Ultra-pure. This analytical procedure gave recoveries of 99.8±3.5～96.8±3.5% in soil and 98.2±4.5～100.8±5.0% in carrot,98.6±5.4～100.3±5.5% in natural pond water for a range of spiked H-9201 concentrations from 0.01 to 1.0 mg/kg.The analytical detection limit in soil, carrot, natural pond water was 0.005,0.008 and 0.005 mg/kg, respectively.LC/IT/MS method development. Detection was performed with an Agilent 1200 MSD ion-trap 6300 system (Agilent, Palo Alto, CA, USA) operating in the electrospray negative mode. The optimized conditions were as followings:nebulizer gas pressure 60 psi, drying nitrogen gas temperature 350℃, drying nitrogen gas flow 9 L/min, vaporizer temperature 400℃, capillary voltage 1200V, the optimized perimeters were obtained with 23.02 mg/L H9201 standard in methanol, and quantization was monitored as [M-H]-ion. The LC-MS mobile phase consisted of 80% phase A (containing acetonitrile and 0.1% formic acid) and 20% Phase B solvent (containing Deion water and 0.1% formic acid) at flow rate of 0.3ml/min.MRM transitions were selected based upon analysis of pure reference standards. The average abundance ratios between the precursors:product ion transitions were determined,317/317 was compared with 317/248.6 and 317/165.7 to ensure the ratios obtained from the plasma standards can be applied to real cases. The recoveries of 89.2±6.8～102.4±4.5% in carrot and 85.5±3.5～105.4±7.5% in soil, 92.5±5.4～100.8±6.0% in natural pond water were archived at the spiked H-9201 concentrations from 0.01 to 1.0 mg/kg.The analytical detection limit in soil, carrot, natural pond water was 0.009,0.009 and 0.007 mg/kg, respectively.3. Dissipation of H9201 in carrot, soil and water after application under field condition.Hydrolysis study of H9201 in different pH water solution. The results of the hydrolysis experiments indicate that hydrolysis of H-9201 started during the 112 days of incubation at pH 7, after this period hydrolysis of H-9201 slowed down, which can be described by modified first-order equation also known as pseudo first-order equation, the half-life was 538.29 days. Dissipation followed the first-order kinetic equation at pH 5 and pH 9, and the hydrolysis rate at pH 9 was faster than at pH 5. The inorganic contents of the natural pond water or biological effects may accelerate the degradation and shorten the half-life, which was 197.2 days. An initial metabolite was found and identified as O-methyl-O-(2,4-dimethyl-6- nitrophenoxy)-N-hydroxy phosphoramide, the most likely pathway of H-9201 hydrolysis is outlined.Dissipation study of H9201 in soil. The results showed that the representation of herbicide persistence by the first-order discontinuous biphasic model was better than simple first-order kinetics and one, two phase exponential decay model, which derived from Microsoft Excel Solver and GraphPad Prism 5.0, and the degradation of H-9201 slowed down from around 30 days after application. The first-order discontinuous biphasic model had a DT50=9.9 days, the best-fit model for H-9201 was finally found to be the first-order biphasic model, and the endpoints derived from this model were accepted for assessment the fate of H-9201, the equation is C=7.0264e-0.0700t1+ 1.1396e-0.0120t2H-9201 residue in post-harvest carrot and in soil. The residue of H-9201 in harvested carrots only came from the environment where the herbicide had been applied before seedling emergence, implying that the residue probably accumulated in carrot via water delivery, since this chemical can persist in the aqueous layer as shown by our hydrolysis study, therefore an environmental impact assessment is needed to determine the bioaccumulation of H-9201 residues in the aqueous environment. The residues of H-9201 present in carrot and soil after application (harvest at 144 days) were less than 0.02 mg/kg in carrot, and less than 0.4 mg/kg in soil.