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1、农药残留检测技术及其应用 从取样到结果的判定,潘灿平 中国农业大学 农业部农产品质量监督检验测试中心（北京） Email： TEL: 010 62733219,2008.1.11 中日韩食品安全分析技术与应用学术报告会,Pesticides are chemicals that we use to kill undesireable organisms. 对农药的认识： 1） 定义的拓展 2） 化学品使用必不可少 3） 安全、合理使用（环境、食品、人）,农业现状,我们生活在一个人口爆炸的时代，对粮食的需求也就不可避免的会很大。WHO预测，2025年全球人口将超过80亿，到2050年将变成10
2、0亿。 同时，人均可耕种的土地将从1960年的1.5公顷减少到2015年的0.2公顷。,农用化学品使用必不可少,病虫草害使得作物减产，其中杂草为1525，病害1550，虫害2035。 如果农民停止使用农药，水果将减产78，蔬菜将减产54，谷物将减产32。 由于作物的病虫草害防治不当，将使产量减少平均达30以上。,农药的使用在现代农业中的地位,由于农药的使用，有效控制了病、虫、草对农作物的为害，使得全世界每年农作物总产量减少3040的损失， 每年挽回经济损失约3000亿美元（USA？）。,生物燃料,专家预测， 在未来50年100年现有的石油燃料将会耗尽。 美国已经颁布总统法令，要求2015年以前
3、实现生物燃料消耗占到总燃料消耗的20。虽然有人声称美国没有足够的土地来实现此目标，然而2007年美国的玉米种植面积还是达到历史新高。按照美国政府的计划，2008年30的玉米将用于发展乙醇工业，在20022003年的水平上增加了50。到2010年，美国的玉米种植面积将扩大至1亿英亩（4200万公顷）。 对生物燃料的需求发展是如此之快，以致很多国家还没来得及准备好应对由于日益减少的农田资源而引发的利益冲突。农民自然愿意种植价格更高的燃料作物而非粮食作物，就如在墨西哥，美国燃料作物需求的增加带动了玉米价格的上升，使得墨西哥的农民更愿意出口他们的农产品而不愿意在本国市场上将其作为粮食产品出售。,美国用
4、于生产生物燃料作物情况,作物保护现状,2006年，全球作物保护市场的总额约为310亿美元， 其中除草剂占46%（142.6亿），杀虫剂占29%（89.9亿），杀菌剂占18（55.8亿），其它为灭鼠剂、植物生长调节剂以及杀软体动物剂（21.7亿）。生物农药占市场份额的1（3.1亿美元），其中90为B.t.（2.79亿美元）。 2006年，Cropnosis估计全球生物技术作物市场销售达到61.5亿美元，使得全球作物保护市场总值达到385亿，其中生物技术作物占市场总额的16%，占全球商业种子市场（300亿美元）的21%。 在61.5亿美元的生物技术作物市场中，转基因大豆的市场份额为26.8亿美元（
5、占总市场份额的44%），转基因玉米为23.9亿美元（占总市场份额的39%），转基因棉花为8.7亿美元（占总市场份额的14%），转基因油菜2.1亿美元（占总市场份额的3%）。,如何正确理解农药残留？,让人们知道食品中含有哪些化学物质，该怎样检测农药含量和哪种情况下是安全的 食物中的天然化学物质有时远比合成化学品危险？,Pesticide residue,“Any specified substance in food, agricultural commodities or animal feed resulting from the use of pesticide. The term inc
6、ludes any derivatives of a pesticide, such as conversion products, metabolites, reaction products and impurities considered to be of toxicological significance.“,注意： 残留的定义很重要， 不同场合要求分析的残留对象可能不同！,CCPR,Determine Priorities,Review Residue Data and,Propose MRLs,Review Toxicology Data,Estimate Short-term
7、 and,Chronic Dietary Exposures,Estimate Values for the ADI,and ARfD,Are the Estimated,Dietary Intakes less than,the Toxicological,Benchmarks?,CCPR,Consider Proposed MRLs in the,context of the Risk Assessment,FAO Residue Experts,WHO Toxicology Experts,JMPR RISK ASSESSMENT,MRL的设定模型,TMDI的计算是根据推荐的MRL值，
8、假设人们每日摄入的食品中都含有MRL水平的农药残留，然后根据人们的膳食结构来计算每日理论摄入量，即：TMDI Fi x Mi 其中Fi 表示平均每天摄入某种食品的量， Mi表示相应食品的MRL。 由于TMDI比较保守，因此WHO在1997年颁布了比较准确的EDI方法来估算人们的每日摄入量， EDI STMRi x Ei x Pi x Fi； 其中， STMRi 是残留中浓度（STMR)。Ei某食品可食部分系数，Pi为食品的加工系数，Fi平均每天摄入某种食品的量。,优良农业措施（GAP）,GAP应包括最佳使用量范围、使用次数、每次使用的间隔期、安全采收期（PHI) 等。 在该过程中，在首先考虑有
9、效性的同时，应同时考虑尽量少施药以减少农药的浪费和对环境的污染。,MRL的主要作用,作为一种法定限量其主要作用是用以检验农作物和食品生产过程是否严格执行优良农业措施，帮助经农药处理的农产品在全球进行进出口贸易。 MRL可保障食品安全？ MRL不是一个安全限量，农业残留超标的食品并不一定必须意味着对人体健康有影响。ADI是通过一系列毒理学研究而得到的一个安全限量，也就是说估计摄入量是否超过由ADI计算的TMDI理论摄入量，才是判断食品是否安全的标准。ADI反应了长期评价；最近还关注急性毒性方面的安全。,Results of Pesticide Residue Monitoring,Results
10、 from the 1989, 1990 & 1991 FDA Total Diet Studies,作物组合? 解决全面制定MRL的钥匙？,将在生物学，分类学，和园艺学特征上接近的作物组合起来，再选出代表作物。,用代表作物的残留数据来建立全组的残留指标。,农药残留分析基本流程,取样、实验室样品、分析部位、提取、样品净化、仪器分析、分析报告、结果报告,Preparation of the analytical sample,美国、日本、德国等多残留分析方法,日本残留農薬多成分一斉分析法展望,LC/MS(SCAN)分析,ENVI-Carb SPE,乙腈提取,ODS SPE,塩析,PSA SPE,
11、GC/MS(SCAN)分析,日本positive list 对应分析方法,(約350農薬),(約100農薬),日本农药多残留分析方法,丙酮提取,ODS净化,CH2Cl2转移,QMA+NH2净化 (SAX+PSA),GC/MS分析,Luke法（FDA),乙腈提取,ODS净化,塩析,HPLC分析,CDFA法,GC/MS分析,乙腈提取,ODS净化,塩析,秋山法,PSA净化,NH2净化,GC-FPD GC-ELCD,Dispersive-SPE,Why use an SPE apparatus for “chemical filtration?” Dispersive-SPE involves the
12、 mixing of the sorbent with the extract in a mini-centrifuge tube to retain matrix interferants, but not analytes.,5/农业行业标准蔬菜、水果中51种农药多残留的测定 气相色谱-质谱法,在重现USDA QuEchERS方法的基础上，结合中国国情扩展农药品种（51种），提高灵敏度，开发适合国情的QuEchERS的GC/MS方法。 重点解决农业部优先监测的13种有机磷（OP）、有机氯（CH）农药的回收率和确证灵敏度问题，进而扩展为51种农药，得到一种简便、灵敏、准确定量的GC/MS确证
13、方法。,NY/T 1380-2007,样品前处理流程图,15g 粉碎 样品+15ml 0.1% 醋酸/乙腈溶液 +6.0g 无水硫酸镁+1.5g 醋酸钠,取2.0ml 上清液至盛有300mg无水硫酸镁100mg PSA和100mgC18离心试管中,5000转/分钟离心1.0分钟,剧烈振荡1.0分钟,旋涡混合器混合1.0分钟,5000转/分钟离心1.0分钟,取1.0ml 上清液至进样器小瓶中，用氮吹仪在室温条件下浓缩至小于0.8ml,分别加入100ul TPP和100ul 分析保护剂混合溶液，用乙睛定容至1.0ml,技术特点： 1. 无水硫酸镁代替无水硫 酸钠和氯化钠脱水 2. 分散固相萃取代替
14、固相 萃取柱净化 3. 添加分析保护剂提高检 测灵敏度,保护剂D05(20mg/ ml) +B06(1.0mg/ ml) 对峰形和峰面积的影响 A: 基质提取+分析保护剂 B: 基质提取 C: 标样+分析保护剂 D: 标样(黄瓜,苹果,韭菜混合基质),A,B,C,D,A,B,C,D,Methamidophos m/z 94,Acephate m/z 136,Omethoate m/z 156,Carbofuran m/z 164,Mevinphos m/z 127,Fenvalerate m/z 167,Leek Matrix,Pest Std+ Protectants,Leek+Pest s
15、td+ Protectants,QUECHERS 方法参考论文,色谱法测定农产品中农药残留时的基质效应，农药学学报，2005年第4期 气质联机分析蔬菜中农药多残留及基质效应的补偿，高等学校化学学报，2006年第2期 应用分析保护剂补偿基质效应与气相色谱质谱快速检测果蔬中农药多残留，分析测试学报，2006年第3期。,California Department of Food and Agriculture Multi-Residue Method,Three PDP laboratories (California, Texas, and Washington) use MRM CDFA met
16、hod： Fifty grams of homogenized sample is extracted with acetonitrile. Sodium chloride is added to the sample/acetonitrile mixture and agitated. All samples are subjected to an initial C-18 solid phase extraction (SPE) clean-up step. The acetonitrile is evaporated from one aliquot of each sample, wh
17、ich is reconstituted with acetone for organophosphate analysis. Additional aliquots are cleaned up using florisil or aminopropyl SPE columns depending on the type of compound or the instrumental analysis to be conducted.,Florida Department of Agriculture and Consumer Services Multi-Residue Method,Th
18、e Florida laboratory uses an acetonitrile-based extraction with aminopropyl solid phase extraction (SPE) cleanup procedures to extract low-fat, high moisture commodities (i.e., fruit and vegetables) for pesticide screening. Fifty grams of homogenized sample is shaken with 50 mL acetonitrile. Sodium
19、chloride is added to the sample/acetonitrile mixture and agitated. Samples for gas chromatography (GC) analyses are cleaned up using an aminopropyl SPE cartridge, eluted with 25% toluene in acetone and reconstituted in iso-octane for instrumental analysis. Samples for liquid chromatography (LC) anal
20、yses are cleaned up using an aminopropyl SPE cartridge, eluted with 3% methanol in acetone and reconstituted in methanol for instrumental analysis. major pesticide classes (e.g., organophosphates, organochlorines, carbamates, triazines, triazoles, pyrethroids, neonicotinyls, strobilurins,Modified Lu
21、ke Multi-Residue Method,One PDP laboratory (Ohio) uses a modified version of the Luke method： 100 g of homogenized sample is extracted with acetone. An aliquot of the filtrate is partitioned with methylene chloride and petroleum ether. Two additional methylene chloride partitions are performed. The
22、final 10 mL extract is reconstituted in acetone and split for separate analysis using various instruments.,New York Modified SPE Method,The New York Department of Agriculture and Markets laboratory uses a method based on the Agriculture and Agri-Food Canada solid phase extraction (SPE) method4 with
23、some improvements based on the Luke extraction. 50g of sample is extracted with 5% ethanol in acetonitrile and 15 grams sodium chloride. The organic layer is dried with sodium sulfate and cleaned up using a combination of SPE cartridges: graphitized carbon (Envi-carb), strong anion exchange (SAX), a
24、nd weak anion exchange (PSA). Compounds are eluted with 3:1 mixture of acetonitrile:toluene, reconstituted in the appropriate solvent, and analyzed by the following instrument/detector combinations: gas chromatography (GC)/flame photometric detector (FPD), GC/tandem mass spectrometry (GC/MS/MS), and
25、 high performance liquid chromatography (HPLC)/tandem mass spectrometry (LC/MS/MS).,Slide from Gnter Lach of Eurofins,Fruits and vegetables,Extraction: Module E 9 Sample weight: 50 g Extraction with ASE GPC with TACS Detection ECD and NPD GC column: DB 5, length 30 m, i. D. 0,25 mm, film thickness 0
26、,25 m,36,Relative frequency distribution of chlorpyrifos residues in apple samples,37,Conclusions,The typical sampling uncertainty For medium size crops with sample size 10 CVS,med = 28% For small crops with sample size 10 CVS,small CVS,med For large crops with sample size 5 CVS,large is about 40% (
27、temporary estimate),Sampling,Consideration of the spread and variability of the residues helps to avoid misleading interpretations of small differences or drawing definite conclusions from a single calculated value. Careful attention to the details of sampling is essential if representative samples
28、are to be obtained.,Preparation of the analytical sample,The preparation of the analytical sample from which the analytical portion is withdrawn consists of two distinct procedures: sample preparation and sample processing. Sample preparation and processing must be carried out according to the aim o
29、f analysis. For instance, to provide data for the estimation of maximum residue limits, the Codex Alimentarius Commission specifies the portion of commodities to which Maximum Residue Limits (MRLs) apply.,Sample preparation,According to the Codex Alimentarius, sample preparation is the procedure use
30、d, if required, to convert the laboratory sample into the analytical sample by removal of parts (soil, stones, bones) not to be analysed. For instance: for lettuce: remove dirty and withered leaves for oranges: remove leaves and stems The method of sample preparation may be the source of substantial
31、 systematic and random errors, which cannot be estimated. However, it is possible to maintain this uncertainty component to a minimum level by strictly following sample preparation procedures, as described in the relevant standard operating procedures (SOPs) attached to the study plan.,Homogenisatio
32、n,Homogenisation implies a mechanical size reduction with suitable equipment, like mincing machines, choppers, blenders, or grinders. If available, it is advisable to add dry-ice during homogenisation in order to improve the efficiency of sample processing, while reducing the risk of thermal degrada
33、tion of pesticides. The homogenisation in the presence of dry-ice produces a fine powder, which is easy to mix,The sampling constant (KS) is the weight (W)of a single increment that must be withdrawn from a well-mixed material to hold the relative sampling (withdrawing and processing) uncertainty to
34、 1% with 68% level of confidence,取样系数与不确定度,Apple samples,Confidence intervals,The uncertainty of the analytical result comprises:,The expression can be modified to incorporate any additional stages,Uncertainty components:,The efficiency and uncertainty of sample processing can be tested utilising th
35、e “sampling constant”, KS,Stability of residues,Systematic studies revealed substantial decrease (40-70%) of the concentration of several analytes (captan, captafol, folpet, chorothalonil, dichlofluanid) during processing at ambient T The extent of loss was dependent on both pesticide and commodity,
36、 and even among varieties Recent study on stability of residues during cryogenic processing : 94 of 106 pesticides tested were stable losses of several pesticides -bitertanol (95%), heptenophos (50%), isophenphos (40%), and tolylfluanid (48%)- reported at Tamb did not occur during cryogenic processi
37、ng Still needs to be demonstrated that has no detrimental effect on some pesticides,Extraction of pesticide residues and metabolites,农药残留分析中包括分离步骤， 即把分析目标物质和干扰物质分离的过程。 多数操作中， 第一步是提取过程。 different extraction liquids (solvents), extraction equipment and the importance of determining extraction efficien
38、cy and method recovery. Filtration and concentration of the sample extracts,标记同位素在分析中的应用,测试的基本步骤： preparation of the samples, homogenization, extraction, clean-up and injection into the adequate equipment for chromatographic separation and measurement. 测定各个步骤的不确定度是GLP要求。 The uncertainty of each step
39、 of the analytical procedure can also be determined. Radiolabeled pesticides can help: 研究回收率和提取效率 check the analytical recovery of residues, 研究残留物质的稳定性 check the stability of residues, and 研究单元操作的不确定度 estimate the uncertainty of processes.,Estimation of the uncertainty of processes,目前还没有一种分析方法， 可以采用
40、一种提取方法， 一种净化过程， 一种检测手段一次分析所有基质类型中的所有可能农药残留。 这样的方法是理想的， 也是不存在的。 -Pan C.,检测与确证手段,GCMS LCMS MSMS SPR？ 快速、无损,incorrectly identified or false positive results,In pesticide analysis, analytes in samples are often incorrectly identified or false positive results are reported. This may be due to: the presen
41、ce of natural co-extractives or other contaminants, which “mimic“ the characteristics of some pesticides, or the lack of proper confirmation of the analyte identity.,Retention time GLC analysis Mass spectrometer HPLC and LC-MS/MS analysis Derivatisation TLC analysis Determination criteria ELISA Deri
42、vatisation,EU对质谱确证方法的要求,全扫描：当用单级质谱记录全扫描图谱时，至少要有四种离子的相对丰度大于等于基峰的10%。如果分子离子峰在参考图谱中的相对丰度10%，则必须包括在内。 选择离子监测质谱方法时要求: 96/23/EC指令附录 I A组 4识别点 96/23/EC指令附录 I B组 3识别点,对质谱确证方法的要求,质量碎片类型和识别点的关系,对质谱确证方法的要求,对于每个诊断离子，信噪比 3:1 测定至少一对离子丰度比 相对离子丰度最大容许偏差 不能超过下表,对其他确证方法的要求,仅对于 B 组物质适用 （限用物质） 对于 A 组物质有1个识别点 LC 或 GC 带 I
43、R检测器 LC带全扫描 UV/VIS检测器 LC带荧光检测器 2-D HPTLC带全扫描 UV/VIS,对其他确证方法的要求,仅对于 B 组物质适用 （限用物质） 对于 A 组物质有1个识别点 GC-ECD (用二根不同极性的色谱柱) LC-酶免测定 (2 种不同的色谱分离或第二种测定方法) LC-带 UV/VIS 单波长 (2 种不同的色谱分离或第二种测定方法),Quality control for pesticide residue analysis,What is it? QC procedures Sample handling Sampling Sample transportat
44、ion Samples at laboratory Sample storage Pesticide standards Stock and working standards Extraction Contamination Interference Control instrumental analysis Dead volume peak shape test Solvent peak shape test System suitability tests (SSTs) Analytical calibration Matrix effects Analytical methods an
45、d recovery determinations Spike or Recovery samples Duplicate analysis Recovery determination Blind samples Proficiency testing Confirmation of results Reporting of results Tools for IQC QC activities,Sample storage,During the storage in the laboratory, the samples must be protected against physical
46、, chemical and mechanical influence, which can lead to changes in the samples and consequently to the analytes. Examples are: air oxidation or other chemical influence, evaporation of water and other volatile components, enzymatic degradation, microbial activity, pollution from the container.,Extrac
47、tion,Analytical portions should be disintegrated thoroughly before or during extraction to maximise extraction efficiency. Over-heating during extraction must be avoided to minimise solvent or pesticide losses. Temperature, pH, etc., must be controlled if they are known to affect extraction efficien
48、cy and/or pesticide stability.,System suitability tests (SSTs),To verify the applicability of the chromatographic system to sample analysis or to reveal degradation in system performance, a system suitability test mixture (SST) is periodically injected in the chromatographic system. The SST mixture
49、contains a series of compounds that have a range of functional groups and selected characteristics. For multiresidue analysis of pesticides the test mixture should contain pesticides covering the retention time range of the compounds that are analysed.,NPD SST,Optimized NPD SST,NPD SST after injection of 40 sample extract,Change of peak shape and RRT,1- peak shape after the system maintenance; 2 after injection of