| Solid phase microextraction (SPME) has been applied for enviormental pollutants analysis with many advantages such as high sensitivity, organic solvent free, etc. For a long time, toxicity risk assessment of pesticide was nearly based on the total pesticide concentration, which neglects the correlation between the total pesticide concentration and the free concentration that interact with target directly. It was mistakenly considered that as long as the total pesticide concentration was determined precisely, toxicology risk assessment of pesticide was reliable, which overestimated the risk. In fact, only freely desolved chemicals are able to go through cell membranes and cause toxic effects. While the bound chemicals will not have the bioavailability by being blocked outside of the cell membranes or target enzyme, due to the large molecular volume or strong polarity. Free concentration is more representative for bioavailability than total concentration, and is a vital parameter for evaluating of bioavailability. Free concentration is related to not only the total concentration of analyte, matrix concentration, but also the binding affinity between target compound and matrix. Human serum albumin (HSA) is the most important abundant transport protein in human plasma. Hence, studying the interaction between pesticide and HSA will contribute to bioavailability assessment in plasma as well as toxicological evaluation. In this work, SPME was appied for studying pesticides residue and interaction between organophosphorus pesticides and HSA. The main results were as follows:1. A SPME method was esteblished for multiresidue analysis in water. A65μm PDMS/DVB fiber was selected coupled with direct immersion sampling mode for analyzing six amide herbicides with GC-MS. And the recovery was80.8%～106.4%with relative standard diviation of2.2%～17.7%. The linear range was0.01～20μg/L (R2>0.991). LOD (limit of detection) and LOQ (limit of quantification) are of0.004～0.02μg/L and0.1～0.6μg/L, respectively. Results showed that the sensitivity of the method fitted the requirement of surface water criteria setted by European Union or World Health Organization.2. A SPME method was developed for analyzing3organophosphorus pesticides in HSA solution. PDMS7μm, PDMS30μm and PDMS30μm fiber were selected respectively for analyzing chlorpyrifos, parathion-methyl and malathion with external calibration approach. The extraction time in phosphate-buffered saline solution was1h for chlorpyrifos,40min for parathion-methyl and10min for malathion. The extraction time in HSA was40min for chlorpyrifos,40min for parathion-methyl and10min for malathion. Results showed that the method has good accuracy and reproducibility.3. The binding constants for OPs-HSA were investigated. The free concentrations obtained by Multiple Standard Solution Methods were adopted for curve fitting with one site saturation mode or two sites saturation mode. The results showed that the binding constants (Ka) abtained by SPME are similar to literature results. The correlation coefficients for curve fitting are greater than0.992. The binding constants for chlorpyrifos, parathion-methyl and malathion are1.41×105,1.45×104and1.07×104at37℃, respectively. Binding constant was greatest for chlorpyrifos, intermediate for parathion-methyl, and lowest for malathion. The results showed higher Kavalue for hydrophobicity compound and also for low temperature.4. The binding force for OPs-HSA was investigated.△H,△S and△G of parathion-methyl and malathion were calculated by Gibbs-Helmholtz equation.△H for two OPs was below zero, which meant binding process was an exothermic reaction. The energy needed for the reaction to occur was less than the total energy released.△S for two OPs was below zero, which meant the system was less chaotic and binding reaction was an entropy reduction process. The△G was below zero, therefore it was a spontaneous reaction for the binding process. The results showed that the binding force for parathion-methyl and malathion were hydrogen bond or Van der Waals’ force.5. The newly developed SPME methods were applied in investigating the OPs’ binding sites on HSA. It was confirmed by site marker competition test. The results showed that parathion-methyl and chlorpyrifos bonded to subdomain IIA, while malathion bonded to subdomain ⅢA. The addition of site markers would increase the binding constant of parathion-methyl and malathion, which indicated a synergetic binding occurred between OPs and site markers.SPME had the significant advantages over current methods:wide applicability, easy operation, high sensitivity, automatic operation ability, etc. It brought new idea for pesticide-protein binding study, and also for competitive/synergetic binding between several ligands with protein. |
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