用于酶热传感器的酶标甲胺磷农药的制备

采用碳二亚胺(EDC)法进行酶标甲胺磷的制备研究。考察甲胺磷和丁二酸酐比例、反应时间对甲胺磷衍生物合成的影响,最终确定二者的最适物质的量比为1:1.5,最适反应时间为15h;甲胺磷衍生物与葡萄糖氧化酶比例对酶标甲胺磷偶联率及酶活的影响实验结果显示,随着甲胺磷衍生物与葡萄糖氧化酶二者比例的增加,酶标农药的偶联率增加,但同时也造成了酶活的降低。考虑到酶标农药还应具备足够的酯酶抑制活性,甲胺磷衍生物与葡萄糖氧化酶二者物质的量比为10:1时制得的酶标农药为最优。     

自制乙酰胆碱酯酶膜对有机磷农药残留的检测研究

首次以自行制备的壳聚糖-乙酰胆碱酯酶膜分别检测了乐果、甲胺磷、敌百虫、敌敌畏、甲基对硫磷5种有机磷农药,发现酶膜对不同类型的有机磷农药的敏感性差异很大,对甲基对硫磷最为敏感,其pI 50为5.57左右,对其他四种农药较不敏感,其pI 50为4左右。以酶活力抑制率20%为农药检出标准,甲基对硫磷与甲胺磷的检出限分别为0.4ppm和1.5 ppm,基本达到实际检测要求。     

臭氧对有机磷农药残留降解效果及检测方法的研究

实验采用臭氧对敌敌畏、甲胺磷、久效磷、乙基对硫磷四种有机磷农药残留进行降解,并分别采用了酶抑制法和气相色谱法对其降解效果进行检测.结果表明,一定浓度的臭氧,作用不同时间,对上述四种有机磷农药残留均有不同程度的降解作用,作用10min即可达到较为理想的降解效果.其中,对甲胺磷降解作用最强为81.72%,敌敌畏57.86%、乙基对硫磷56.78%、久效磷47.53%.     

酶标甲胺磷交联反应的pH及缓冲体系优化

采用碳二亚胺法制备酶标农药(过氧化氢酶标记甲胺磷),以酶标农药对鸡肝酯酶的抑制率和抑制后酶标农药-酯酶复合物中标记酶酶活为指标,对制备酶标甲胺磷交联反应的pH和所用缓冲液类型进行优化,并对其偶联率进行了分析。结果发现,pH增加有效促进了甲胺磷和标记酶分子之间的交联,而缓冲体系类型对此交联反应的影响相对较小。然而随之持续上升的偶联率带来的空间位阻效应也逐渐明显。随着pH增加,标记物抑制率和抑制后的标记酶活均呈现先增加后略下降的趋势,且分别在pH为6.0和5.5时达到峰值,其峰值时偶联率分别为24和22。综合考量,采用pH 6.0的2-(N-吗啉)乙磺酸一水合物(MES)缓冲液制备的酶标农药,其抑制率和标记酶活均达到较优值,分别为45.29%和47.67U,此时其偶联率约为24。     

茶叶中甲胺磷农药快速测定研究

利用有机磷农药能抑制植物酯酶酶促反应的机理,建立了快速测定茶叶中甲胺磷农药残留的方法。用70%饱和硫酸铵对面粉中植物酯酶进行纯化,在45℃、pH4.5～5.5时制备的酶催化反应活性最大,酶催化反应动力学显示10min内反应呈线性关系。测定甲胺磷农药残留反应体系最大吸收峰是在480nm之间,最适乙酸-β-萘酯用量为1.0ml;在甲胺磷浓度在0.1～1.0mg/kg之间时,反应体系的吸光值与甲胺磷浓度呈线性相关;在茶叶汁提取液回收率为84%。     

建立一种增强酶抑制法快速检测谷物中有机磷农药残留

本研究的目的是建立一种增强的酶抑制法,实现对谷物中有机磷农药残留的快速检测。通过对酶抑制法中所使用的提取剂、酶、增强剂的选择和优化,并比对抑制率的差异,最终确定使用丁酰胆碱酯酶作为催化酶、乙醇作为提取剂、溴代琥珀酰亚胺作为增强剂。在此条件下,建立了硫代磷酸脂类有机磷农药对硫磷、辛硫磷、毒死蜱和三唑磷的标准曲线(决定系数R2 > 0.98),抑制中浓度(IC₅₀)分别为0.01、0.11、0.16和0.03 μg/L,满足了硫代磷酸酯类农药在不同谷物中的安全残留限量的检测需求。采用本研究改良的酶抑制法对200个不同来源的谷物样本(稻谷、小麦、玉米、糙米、杂粮)进行分析,检测出13个阳性样本,同时,因酶抑制法特异性的限制,产生了1个假阳性和1个假阴性样本。检测结果与气相色谱法符合率达到98%,表明该方法可有效用于谷物中有机磷农药残留检测。本研究可为我国粮食质量安全监管提供有力的技术支撑。     

产有机磷农药降解酶菌株的筛选及酶学性质研究

通过用乙酰胆碱酯酶抑制法检测有机磷农药,从土壤中分离筛选出能降解敌敌畏的7个菌株,从其中降解能力较强的菌株OPD7提取胞内酶,并进行酶学性质的研究。结果表明,该酶分解敌敌畏的最适pH为7.0,最适温度为42℃,pH稳定范围为6-8,在低于47℃时稳定;该酶也能降解乙酰甲胺磷、甲胺磷、乐果和氧化乐果。     

试纸法快速检测果蔬中有机磷及氨基甲酸酯类农药残留

为了快速检测果蔬中的农药残留,研制基于酶抑制法的农药残留速测试纸。该试纸以鸭血浆胆碱酯酶为检测用酶,2,6-二氯靛酚乙酸酯为底物。反应后根据颜色变化目测判断农药残留情况,强阳性、弱阳性和阴性的颜色分别为白色、浅蓝色和蓝色。用该法检测只需13min,对敌敌畏的敏感性最强,对甲胺磷的敏感性较差,对几种常见的有机磷及氨基甲酸酯类农药的检出限在0.001～6mg/kg之间。该法简便、快速,成本低,适合果蔬中部分农药残留的现场监督检测。     

高通量酶抑制法快速测定蔬菜中有机磷和氨基甲酸酯类农药残留

目的 建立一种室温下高通量酶抑制法快速测定蔬菜中农药残留的方法。方法 从酶抑制法检测原理出发,以阳性蔬菜样品为研究对象,通过对提取剂、提取时间及反应体系的选择和优化,确立酶反应体系。采用该方法检测市售蔬菜样品,对筛选出阳性样品进行定量检测复核,验证方法有效性。结果 蔬菜样品用乙腈作为提取液,采用超声提取,上清液氮吹后加入磷酸盐缓冲溶液(pH 8.0)作为最终样液,农药提取效率最高,且不会对酶抑制法反应体系造成影响。采用优化后的高通量农药速测方法,以室温(25±5)℃下酶抑制率≥40%作为阳性判定标准,对筛选出的734个阳性样品进行仪器定量分析复核,检出符合率达到92.1%。结论 该方法有效提升了农药提取效率和检测效率,可有效用于蔬菜中有机磷和氨基甲酸酯类农药残留的快速检测。     

蔬菜有机磷农药残留快速检测方法的应用

以黄瓜为试材，采用酶抑制半定量快速检测方法分析几种常见有机磷农药的残留动态，结果表明，敌敌畏和辛硫磷的降速度最快，氧化乐果和甲基对硫鳞次之，甲胺磷的降解速度最慢，喷洒后5d仅有约一半降解，市场抽样检测表明，市场上销售的果菜类蔬菜均受到不同程度的有机磷农药污染，其中以黄瓜最为严重。     

气相色谱-串联质谱内标法测定违规添加的有机磷类农药

目的建立气相色谱-串联质谱内标法定量测定7种有机磷类高毒农药的分析方法。方法采用电子轰击离子源模式电离,使用TG-1701 MS毛细管色谱柱,邻苯二甲酸二戊酯做内标物,测定农药试剂中甲胺磷、甲拌磷、特丁硫磷、氧乐果、久效磷、甲基对硫磷、对硫磷的含量。结果 7种有机磷农药的相对标准偏差为2.1%~3.5%,变异系数2.0%~3.4%,3个质量水平的样品加标回收率范围为98.6%~102.3%,线性相关系数均大于0.999。结论该方法简便、快捷、有效地分离了样品中7种高毒有机磷农药,而且精密度和准确度高、线性关系好,适用于有机磷农药的测定。     

娃娃菜农残消降组合方法及对品质的影响

以娃娃菜为试验对象,研究了有机磷水解酶、吐温、臭氧3种处理方法及其两两组合方法对蔬菜中7种有机磷农药残留的消降效果及对蔬菜营养、色泽和质地的影响。结果表明:6种消降方法都能有效降解娃娃菜表面的残留农药,吐温+臭氧、吐温+酶制剂组合方法较单一消降方法有明显的增效作用。吐温+臭氧组合处理消降率达65%～81%,吐温+酶制剂组合处理敌敌畏、毒死蜱、马拉硫磷、甲基对硫磷的消降率达65%～99%,但对甲胺磷、乙酰甲胺磷、氧乐果等内吸性农药的消降率只有40%。蔬菜经消降技术处理后,L*值、a*值、b*值与对照相比没有明显差异,还原型Vc仍保留在80%以上,且能保持其固有的感官质量要求。     

Use of a new rapid bioluminescence method for screening organophosphate and N-Methylcarbamate insecticides in processed baby foods.

An enzyme with high specific affinity for organophosphate and N-methylcarbamate insecticides has been incorporated into a new test for detection of these insecticides at the level of parts per billion (ppb) (commercially available as the Charm Pesticide Test). To measure the extent of insecticide inhibition of the enzyme, a specific bioluminescent substrate is used. The signal is counterproportional to the amount of insecticides. Random sampling of four baby food brands and testing for the cumulative levels of organophosphate and N-methylcarbamate insecticides found carbaryl to be the most common residue. Out of the 155 samples tested there were 132 negative samples (85.2%) and 23 suspected positive samples (14.2%). The suspected positive samples were further analyzed by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Carbaryl was confirmed in 18 of the samples. One of the samples contained an active metabolite of tetrachlorvinphos and in 3 of the positive samples an insecticide could not be identified by GC/MS. One positive sample was not processed for confirmation due to high fat content.     

Recoveries of four representative organophosphorus pesticides from 18 plant products belonging to different botanical categories: implications for matrix effects.

A study of the matrix effect was performed in order to evaluate the influence of some matrices on the gas chromatographic responses of representative organophosphorus insecticides. In particular, three fortification levels and the maximum residual level of four organophosphorus pesticide standard solutions were added into 18 matrices (15 belonging to five specific botanical categories and three botanically unclassified). The recoveries of the examined pesticides were determined using a simple official multiresidue method of detection, without any additional clean-up step. Recoveries were estimated based on standards prepared in solvent. The recovery percentages of the most polar pesticides, especially methamidophos (20.2-288.4%), were much higher than those of non-polar ones (chlorpyrifos and methidathion 25.7-136.4 and 37.6-292.6%, respectively), except for quince. Pronounced matrix effects (>120%) were observed to the lowest fortification levels (maximum residual levels established by the European Union), of dimethoate and methamidophos (i.e. the most polar analytes) in the majority of product extracts. Furthermore, it was proved that there is no correlation between the classification of plant products, according to their botanical characteristics, and the recoveries of pesticides in the equivalent extracts. Indeed, there were significant differences in the recoveries of pesticides in extracts, derived from matrices belonging to the same botanical group (especially in the categories of pome fruits and citrus). Consequently, it was shown that, under the examined conditions, obtaining recovery data, derived from only one representative matrix, with the purpose to validate an official method in its botanical category is, possibly, an erroneous practice.     

Organophosphorus pesticide residues in milled rice ( Oryza sativa ) on the Chinese market and dietary risk assessment

The present study investigates the occurrence of acetylcholinesterase (AChE)-inhibiting organophosphorus (OP) pesticide residues in milled rice samples obtained form local markets in China during the period 2004–2006 and estimates their cumulative exposure. Concentrations of OP pesticides were determined by gas chromatography with flame photometric detection (GC–FPD). The results showed that 9.3% of the samples contained detectable residues of at least one of the seven target OP pesticides (chlorpyrifos, dichlorvos, omethoate, methamidophos, parathion-methyl, parathion and triazophos) mainly used for agriculture in China, with concentrations ranging 0.011–1.756 mg kg^?1. Rice consumption data was obtained from an individual food consumption survey. Relative potency factors (RPFs) for each pesticide were calculated with methamidophos as the index compound (IC), using 1- or 2-year chronic non-observed adverse effect levels (NOAEL) for AChE inhibition, mostly in rat brain, obtained from international evaluations of pesticides. Exposure to AChE-inhibiting pesticides for the population above 7 years old at P99.9 represented 52–94.5% of the acceptable daily intake (ADI) expressed as methamidophos. Estimated exposure for children aged 2–4 and 4–7 years at P99.9 were 119 and 104.3% of the ADI level, respectively. This study suggests that a yearly monitoring program for OP pesticide residues and strict implementation of the national safety standard for milled rice is necessary.     

烟田常用杀虫剂对烟蚜茧蜂的毒力及其保护酶的影响

为了筛选对烟蚜茧蜂安全性高的药剂并明确烟田常用杀虫剂对烟蚜茧蜂保护酶的影响,测定了3种烟田常用杀虫剂对烟蚜茧蜂成虫的毒力以及不同浓度的吡虫啉对烟蚜茧蜂成虫体内超氧化物歧化酶、过氧化物酶和过氧化氢酶活性的影响。结果表明,杀虫剂对烟蚜茧蜂成虫的毒力大小顺序为:啶虫脒 > 高效氯氟氰菊酯 > 吡虫啉;从不同浓度的吡虫啉对烟蚜茧蜂生命活动和防御机制的影响来看,除LC₁₀浓度吡虫啉促进烟蚜茧蜂成虫体内POD活性提高和3个浓度处理后12 h成虫CAT活性显著提高外,其余处理浓度和时间对3种酶的活性呈现抑制作用。此结果为探明烟蚜茧蜂对杀虫剂的防御机制提供了一定的理论依据。     

气相色谱（GC）法测定蔬菜中甲胺磷的含量

采用气相色谱测定农产品中甲胺磷的含量。结果显示：甲胺磷在0.004μg/mL~0.04μg/mL范围内,线性关系良好（r=0.999 8）,平均回收率为99.41%,平均RSD为1.09%。该法简便、快速、准确,可为农产品中甲胺磷控制标准的制定提供依据。     

2001年我国部分蔬菜和肉类污染状况调查及分析

为了解我国蔬菜和肉类污染的状况 ,受卫生部和国家三绿工程办公室委托 ,我们于 2 0 0 1年秋季在全国 14个城市采集了 6种蔬菜 10 8份样品和 30份猪肉及 15份鸡肉进行了有关污染物的污染状况调查。监测指标有铅、砷、甲胺磷和氯霉素等。结果发现所检的 4 5份肉的铅、砷和氯霉素全部符合国家标准 ,10 8份蔬菜中有一份铅超标 ,砷含量全部合格 ,但是发现蔬菜中存在滥用剧毒农药甲胺磷的情况 ,蔬菜中甲胺磷的平均检出率为 12 % ,检出率最高的为卷心菜 2 7 7%。     

Dietary risk assessment of organophosphorus and dithiocarbamate pesticides in a total diet study at a Brazilian university restaurant

In this study ready-to-eat food samples were collected in the production line of the university restaurant of the University of Brasilia, Brazil, which serves non-vegetarian and vegetarian meals daily. Samples were analysed for the presence of ten organophosphorus insecticides (OPs) by GC/FPD, after extraction with ethyl acetate and anhydrous sodium sulfate (LOQ = 0.002 mg kg(-1)), and for dithiocarbamate fungicides, as CS(2), using the spectrophotometric method (LOQ = 0.05 mg kg(-1)). About 43% of the 175 samples analysed contained at least one OP compound at levels up to 1.83 mg kg(-1). Methamidophos was the compound most detected (37.7%), present in most of the soup, soybean and salad samples. No OP residues were found in fruit juice, beans and bran rice samples. The cumulative acute intake of OPs was estimated using methamidophos and acephate as index compounds (IC). The total cumulative intake represented 9.1% and 47.7% of the methamidophos ARfD for the non-vegetarian and vegetarian diets, respectively. When acephate was used as IC, the total intakes represented 20.7% and 116% of the ARfD for the non-vegetarian and vegetarian diets, respectively. Dithiocarbamates were detected in 70% of the 177 samples analysed, at levels up to 0.51 mg kg(-1) CS(2); all salad samples were positive and no residues were found in fruit juice. The chronic intake of dithiocarbamates represented 8.6 and 8.9% of the ADI (mancozeb) for the vegetarian and non vegetarian diets, respectively.