^(14)C-Carbofuran在土壤及玉米中的传导与分布

以14C－carbofuran作为示踪剂，研究其在土壤中的稳定性及其在玉米中的分布和传导，14C－carbofuran在土壤中有很好的稳定性，具有向顶传导性，向地上部分传导，最终积聚到植株的叶部或叶路部位，老叶比新叶多，叶缘高于其它部位，根上部比根下部含量高。

Investigation of the interaction of carbofuran and DNA by voltammetry and fluorescence with the aid of chemometrics

The interactions of carbofuran and DNA were studied using voltammetry and fluorescence spectroscopy.The formation of carbofuran-DNA makes the current peak of DNA decreased by voltammetry method.The binding number（n） and constant（Ka） for complex carbofuran-DNA were calculated to be 1.06±0.04 and 0.11±0.03mol^-1 L,respectively by fluorescence measurement.Chemometrics approach,such as singular value decomposition（SVD） was used to evaluate the number of spectral species in the drug-DNA binding process.And the pure spectra and concentration profiles in the kinetic system were clearly deduced by multivariate curve resolution alternating least squares（MCR-ALS） with the initial estimates by evolving factor analysis（EFA）.

Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people＇s health and environmental protection, the hapten 4-[[（2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy） carbonyl]- amino]-butanoic acid （BFNB） of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier （BFNB-bovine serum albumin, BFNB-BSA） conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay （ELISA）, based on BFNB-ovoalbumin conjugates （BFNB-OVA）. Purified monoclonal antibody （McAb） was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line （5D3） secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1：2.048 × 10^3 and 1：1.024 × 10^6, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 10×9 L mol^-1, with an IC50 value of 1.18 ng mL^-1 and a detection limit of 0.01 ng mL^-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized （4.59 × 10^-4% for 2,3-dihydro-2,2- dimethyl-7-benzofuranol and less than 3.0 × 10^4% for others）. The prepared McAb had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of carbofuran.

The Separation of Catechol from Carbofuran Phenol by Extractive Distillation

In this study, extractive distillation has been applied to separate catechol （CAT） from carbofuran phenol （CFP） with high purity and yield. The relative volatility of CFP to CAT was measured, and the choice of separating agents was investigated. The experimental results indicated that CFP/CAT is an azeotropic system with an azeotropic point at 93.40℃/0.400 kPa and an azeotropic mixture containing 49.96% of CFP and 50.04% of CAT. Data from the determination of the relative volatility have shown that separating agents such as diglycol and 4-butylcatechol （4-TBC） are able to increase the relative volatility up to 1.90. In one shot process batch extractive distillation of CFP mixture with 3% （by mass） diglycol as separating agent, the purity and yield of the obtained CFP was 99.0% and 95.0%, respectively, while the distillation without separating agent provided a purity and yield of only 98.0% and 90.0%, respectively, There was no residual separating agent found in the product.

Effect of Ionic Liquid on Detection of Carbofuran by Plant-lipases Inactivation

Ionic liquids （ILs） as additive in phosphate buffer for detection of carbofuran by plant-lipases inhibition method is described. The higher inhibition efficiency and the shorter analysis time can be obtained by using N-butylpyridinium tetrafluoroborate ionic liquid-phosphate buffer mixtures instead of pure phosphate buffer.

STUDY ON RESIDUES OF ^(14)C-CARBOFURAN IN MODEL RICE-FISH ECOSYSTEM

Residues of 14C- carbofuran were studied in model late- rice ecosystem (LRE) and early- rice ecosystem (ERE). The treatment consisted of two rates of the pesticide (1x) and 2.5x). At day 56 after application, 7.3% (1x) and 2.9% (2.5x) of the pesticide and its degradative products remained in the water of the LRE, and 1.8% (1x) and 2.4% (2.5x) of them remained in the water of the ERE. At harvest, 37.5% (LRE) and 24.0% (ERE) of the pesticide applied were detected in the upper layer of the soil; and 40.6% (LRE) and 26.9% (ERE) remained in the lower layer of the soil. The residues in the rice plants increased at the first stage, reached maximum levels during day 14 to 28 after application, and decreased thereafter. At harvest, residues in the stems and leaves in the two treatments (1x and 2.5x) were 3.91μ g/g and 7.78μ g/g (LRE) and 5.04 μg/g and 17.29 μ g/g (ERE) respectively. Residues in the ears were about 1/8 to 1/12 of that in the other parts of the plants. The pesticide residues in fish bodies in both experiments were also determined.

Oxidation Degradation of Aqueous Carbofuran Induced by Low Temperature Plasma

The oxidative degradation of aqueous carbofuran, a heavily used toxic carbamate insecticide by low temperature plasma, was investigated. The results show that the treatment efficiency increases with the increase in initial concentration. Raising the treatment temperature and changing the pH value can result in enhanced degradation of carbofuran in solution. The results also show that low temperature plasma treatment can effectively remove chemical oxygen demand （COD） of carbofuran in the solution.

Interaction of Carbofuran and Bovine Serum Albumin by Spectrometry

[Objective]The aim was to study the interaction characteristic of bovine serum albumin （BSA） and carbofuran. [ Method]With synchronous fluorescence spectrometry adopted, the interaction of carbofuran and BSA in Tris-HCI buffer system （pH 7.40） was investigated. The binding constants at different temperatures were calculated and the interaction types between carbofuran and BSA were discussed. [ Result] Under normal physiological conditions, higher quenching effect of carbofuran on BSA was electrostatic interaction. The changes of different drug concentrations and temperature proved a static quenching of carbofuran with BSA. The binding constants （KSV） at 25 ℃, 37 ℃ and 50 ℃ were 1.17 × 10^4, 1.07 × 10^4 and 0. 99 × 10^4 L/mol respectively with ratio of carbofuran and BSA at 1 ： 1. [ Conclusion ] The research is of guiding significance for learning transport and metabolism of carbofuran at molecular level.

Sub-acute Toxicity of Carbofuran on Acetylcholinesterase Activity in the Freshwater Catfish,Clarias batrachus

The inhibition of acetylcholinesterase (ACHE) activity has been widely used as a biomarker in an animal exposed to the pesticides. However, the interaction of extensively used organocarbamate insecticide, carbofuran, with the nervous system of the aquatic organisms is not properly studied. AChE is a key enzyme which catalyses the hydrolysis of acetylcholine, a neurotransmitter at the neuromuscular junctions, and thus regulates the neurotransmission system. In the present study, we have evaluated the impact of sub-acute concentrations (0.01 and 0.02 mg/L i.e. 1/20th and 1/10th of LC50) of carbofuran on the activity of acetylcholinesterase,from different tissues of Clarias batrachus, a fresh water teleost, after 96 hr and 15 days exposure periods in vivo. The carbofuran significantly reduced the activity of AChE in different tissues of C. batrachus at both concentrations and periods of exposure. The greater inhibition of AChE activities were recorded in fish tissues at higher carbofuran concentration (0.02 mg/L) after longer (15days) treatment period. The inhibition of AChE activity in all fish tissues tested was dependent on pesticide concentration and the duration of treatment. AChE from the tissues of C. batrachus was found to be a true cholinesterase as it was completely inhibited by the small concentration (nM) of eserine as tested in vitro. It was found that carbofuran at very low concentration exerted significant inhibitory effect on AChE activity in fish tissues.

胶体金免疫层析法快速检测蔬菜中3种农药残留

基于胶体金免疫层析原理研制吡虫啉、丙溴磷及克百威快速检测试纸条,评价其在大蒜、白菜检测中的应用效果。采用柠檬酸三钠还原法制备胶体金,用胶体金颗粒标记吡虫啉、丙溴磷及克百威单克隆抗体作为检测探针,以硝酸纤维素膜为固相载体,包被吡虫啉-BSA、丙溴磷-BSA及克百威-BSA偶联物为检测线,羊抗鼠IgG抗体为质控线,制备胶体金免疫层析试纸条,并对金标抗体用量、标记抗体浓度、检测线包被浓度等参数条件进行优化,评价试纸条的灵敏度、特异性、稳定性和准确性。以大蒜、白菜为对象,优化了样品前处理方法,并对这2种蔬菜样品进行阴性检测及添加标准品农药检测。结果表明,优化后的工艺参数中吡虫啉、丙溴磷、克百威的最适标记抗体质量浓度分别为9.6、8.4、9.6μg/mL,抗原的包被质量浓度均是0.3 mg/mL,检测用时约15 min。吡虫啉、丙溴磷及克百威的检出限分别为20、3000、20 ng/mL,符合国标对2种蔬菜的限量规定。检测与吡虫啉、丙溴磷及克百威功能和结构相似的其他药物时无交叉反应,特异性较好。克百威和吡虫啉农药的最适提取剂分别为4 mL乙酸乙酯和4 mL甲醇,样品用量均为2 g,上清液3 mL;丙溴磷农药的最适提取剂是9 mL乙酸乙酯,样品用量3 g,上清液8 mL,上清液用氮气吹干。50份蔬菜阴性样品和加标样品的检测结果显示,假阳性率为6%,假阴性率为0。可见,研制的试纸条适用于大蒜、白菜现场快速检测以及基层实验室中大量样本的筛查,可为大蒜、白菜中农药残留控制提供有效监管手段。

克百威纳米抗体的结构解析与体外进化研究

克百威曾是一种广泛用于农业虫害防治领域的氨基甲酸酯类农药,由于其对哺乳动物具有高毒性,对人畜安全及环境造成较大威胁,因此各国陆续出台了克百威禁限用政策,但其在农产品中被检出超标的问题仍时有报道,故对其加强监测十分必要。纳米抗体是一种新型的基因工程片段抗体,具有高稳定性、结构简单和高表达量等优势。本研究通过体外亲和力成熟技术,对实验室已有的克百威纳米抗体Nb5进行改造,进一步提升其灵敏度。首先,基于AlphaFold系统,预测纳米抗体三维结构并验证模型合理性;以分子对接结合丙氨酸扫描技术,研究克百威与纳米抗体的识别机制,并验证关键结合位点。结果表明:克百威分子进入了由CDR2、CDR3及FR3区组成的活性口袋腔,纳米抗体通过氢键和疏水作用力识别克百威,所涉及的关键氨基酸分别为Arg56、Trp58、Thr101、Val102、Ala103、Asp104和Cys105。基于噬菌体展示技术,构建了克百威纳米抗体的定点饱和突变库,并筛选到一株突变体H10,其IC_(50)值为17.6 ng/mL,其灵敏度比野生型Nb5提升了1倍,亲和力常数为1.54×10^(6)L/mol。该研究结果为开发针对小分子药物的纳米抗体体外亲和力成熟技术提供了一种有效的新思路。

气相色谱-质谱法快速筛查食物中毒物质克百威

目的通过气相色谱-质谱法对急性克百威食物中毒事件进行快速检测。方法17份可疑样品经乙酸乙酯快速萃取,离心后取上清液,Rxi-5ms色谱柱(30 m×0.25 mm,0.25μm)分离,应用全扫描模式(full scan,Scan)和谱库检索定性中毒物质,通过QuEChERS法前处理和选择离子扫描模式(selective ion monitoring,SIM),外标法定量。结果在凉粉残留物、1份洗胃液、1份血液及2份不明白色粉末样品中均检出氨基甲酸酯类农药克百威及其中间体呋喃酚,其他样品均未检出,凉粉残留物中克百威质量浓度为253.0μg/mL,呋喃酚质量浓度为546.6μg/mL。克百威及呋喃酚在0~5.0μg/mL范围内线性关系良好(r>0.999),检出限(limits of detection,LODs)分别为0.0014~0.0344μg/mL、0.0016~0.0217μg/mL,定量限(limits of quantitation,LOQs)分别为0.0048~0.1146μg/mL、0.0053~0.0722μg/mL,相对标准偏差(relative standard deviations,RSDs)分别为4.6%~6.6%、3.5%~7.6%。在0.25、0.50、1.00μg/mL3个浓度水平的平均加标回收率为81.4%~107.1%,RSDs为1.9%~7.6%。结论此方法操作简单、灵敏度高、稳定性好,为急性食物中毒事件提供了快速、高效的检测方法。

Photocatalytic degradation of carbofuran in TiO_2 aqueous solution: Kinetics using design of experiments and mechanism by HPLC/MS/MS

The photocatalytic degradation kinetics of carbofuran was optimized by central composite design based on response surface methodology for the first time. Three variables, TiO2 concentration, initial pH value and the concentration of carbofuran, were selected to determine the dependence of degradation efficiencies on independent variables. Response surface methodology modeling results indicated that the degradation efficiency of carbofuran was highly affected by the initial pH value and the concentration of carbofuran. Then nine degradation intermediates were detected by HPLC/MS/MS. The Frontier Electron Densities of carbofuran were calculated to predict the active sites on carbofuran attacked by hydroxyl radicals and photoholes. Point charges were used to elucidate the chemisorption pattern on TiO2 catalysts during the photocatalytic process. By combining the experimental results and calculation data, the photocatalytic degradation pathways of carbofuran were proposed, including the addition of hydroxyl radicals and the cleavage of the carbamate side chain.

Electrochemiluminescence Behavior of Ru（bpy）3^2＋/ Carbofuran System on an Electrically Heated Microelectrode Chip

An electrically heated microelectrode chip （HMEC） was designed and the Ru（bpy）3^2＋/carbofuran electro- chemiluminescence （ECL） systems were applied to characterize the performance of the HMEC. The ECL intensities increase at elevated electrode temperature, and the detection limit at 60℃ （electrode surface temperature） was about 10 times lower than that at 30℃. The results indicate that new heated electrode can be handled easily and can be mass produced, the difference between the electrodes was little. The stability of the HMEC was good since the electrode surface can hardly be destroyed during detection and storage.

不同保存条件下呋喃丹标准储备液稳定性研究

研究呋喃丹标准储备液在不同保存条件下稳定性。将呋喃丹标准品配制成浓度为0.1μg/mL、0.2μg/mL、8.0μg/mL的标准品储备液,贮藏在20℃、4℃、-20℃条件下,分别于保存0 d、10 d、20 d、34 d取上述样品,多反应离子检测(MRM),气相色谱-质谱联用(GC-MS/MS)法检测其呋喃丹含量。呋喃丹的标准储备液在20℃、4℃和-20℃保存条件下均可发生分解,-20℃条件下分解速度最慢,4℃次之,20℃分解速度最快,高浓度较低浓度分解速度慢。呋喃丹标准储备液应在-20℃且高浓度条件下保存,且得到不同温度不同浓度保存条件下呋喃丹标准储备液存放的有效期,为呋喃丹中毒案件的法医学鉴定中标准溶液保存和检测结果的可靠性提供实验依据。

对比教学法在大型仪器实验教学中的应用——以番茄汁中克百威的检测为例

以番茄汁中克百威的检测为例,将对比教学法应用于高效液相色谱仪(HPLC)和液相色谱-质谱联用仪(HPLC-MS)的实验教学中。在理论教学部分,对比两种大型仪器的原理、构造、应用特点及其在克百威检测中的异同。在上机实操部分,由于番茄汁中的克百威含量低于HPLC法的检出限但高于HPLC-MS法的检出限,两种仪器得出截然不同的检测结果。对比教学法切实提高了实验教学效率,提升了学生的大型仪器综合运用能力,对高校应用型人才的高质量培养具有重要意义。

DNA damaging effects of carbofuran and its main metabolites on mice by micronucleus test and single cell gel electrophoresis

The DNA damaging effects of the carbamate pesticide carbofuran and its four metabolites (carbofuranphenol, 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran) on mice were evaluated by single cell gel electrophoresis (SCGE) assay and micronucleus test. KM mice were exposed to test compounds with different doses of 0.1, 0.2 and 0.4mg/kg through intraperitoneal injection two times with an internal of 24 h, and then killed by cervical dislocation 6 h after the second injection. In SCGE assay, isolated mice peripheral blood lymphocytes were employed to determine DNA damaging degree after a 1 h treatment by test compounds and a following electrophoresis. Carbofuran and carbofuranphenol showed negative results in both test and had no obvious toxicity. 3-hydrocarbofuran and nitrosocarbofuran were positive.3-ketocarbofuran could not induce micronucleus formation but caused significant DNA migration in SCGE test. These tests revealed that 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran are potential mutagesis and further research is needed.

基于离子液体原位泡腾萃取法在丹参及西洋参克百威残留分析中的应用

本研究建立了一种新颖的基于酸性离子液体的原位泡腾萃取技术的前处理方法。酸性离子液体1-丁基-3-甲基咪唑硫酸氢盐呈酸性,能与碳酸氢盐反应产生二氧化碳,起到泡腾萃取的效果,它还能与六氟磷酸铵发生离子交换作用而置换成疏水性离子液体1-丁基-3-甲基咪唑六氟磷酸盐,使其与水相分离,通过离心获取萃取相,最后通过高效液相色谱仪测定萃取相中农药的含量。考察了酸性离子液体、碳酸氢钠和氯化钠用量对萃取效率的影响,最终所得最佳条件为200 mg酸性离子液体、0.4 g碳酸氢钠和1.0 g氯化钠。在最优条件下,克百威的线性范围为0.3~4.0μg/mL,日内和日间的相对偏差(RSD)为4.6%和10.2%,丹参和西洋参在4.0μg/mL加标水平下,加标回收率为94.7%(西洋参)和74.6%(丹参),将此方法可用于不同基质中药材和饮片中农药残留的处理。

液相色谱-质谱联用法测定韭菜中克百威残留量的不确定度评定

目的:采用液相色谱-质谱联用法测定韭菜中克百威残留量,并对测定过程的不确定度进行评估。方法:采用《食品安全国家标准 植物源性食品中331种农药及其代谢物残留量的测定液相色谱-质谱联用法》(GB 23200.121—2021)中的方法测定韭菜中克百威残留量,依据《化学分析中测量不确定度评估指南》(RB/T 030—2020)和《测量不确定度评定与表示》(JJF 1059.1—2012)对测量不确定度进行评定。结果:对检测过程中各不确定度分量进行计算与合成,韭菜中克百威残留量可表示为(0.020±0.002)mg·kg^(-1)。在本实验室条件下,标准溶液配制过程和标准曲线拟合对韭菜中克百威残留量测量不确定度的贡献较大,标准溶液定值准确性、测量重复性、提取溶液量取、样品称量以及回收率的贡献较小。结论:研究结果对实际操作中减少不确定度、了解测量结果的置信水平和离散程度具有重要的参考意义。

HPLC-MS法测定茯苓中4种氨基甲酸酯类农药残留

目的:建立Qu ECh ERS样品前处理结合高效液相色谱-串联质谱法检测茯苓中氨基甲酸酯类农药的检测方法。方法:茯苓样品用含1%醋酸的乙腈溶液提取,盐析剂盐析后加入Mg SO4、PSA和C18进行萃取、净化,高效液相色谱-串联质谱仪检测,根据色谱峰的保留时间及特征离子对定性,基质匹配标准曲线外标法定量。结果:在2.0~100.0μg·L^(-1)时,各目标物线性关系良好,相关系数R^(2)>0.998;在0.015 mg·kg^(-1)、0.060 mg·kg^(-1)和0.600 mg·kg^(-1)添加水平下,回收率在77.3%~117.2%,相对标准偏差为0.2%~3.1%(n=6);方法检出限在0.60~1.00μg·kg^(-1)。结论:该方法准确度高、灵敏度好、高效快速,可用于茯苓中4种氨基甲酸酯类农药残留量的质量控制。