北京－食品安全中日韩论坛－－潘灿平：农药残留检测技术及其应用－－从取样到结果的判定课件

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 627332192008.1.11 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100亿。同时，人均可耕种的土

2、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。 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

3、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。 美国用于生产生物燃料作物情况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 residueAny specifi

4、ed substance in food, agricultural commodities or animal feed resulting from the use of pesticide.The term includes 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

5、不同！CCPRDetermine PrioritiesReview Residue Data and Propose MRLsReview Toxicology DataEstimate Short-term and Chronic Dietary ExposuresEstimate Values for the ADI and ARfDAre the Estimated Dietary Intakes less than the Toxicological Benchmarks?CCPRConsider Proposed MRLs in the context of the Risk Ass

6、essmentFAO Residue ExpertsWHO Toxicology ExpertsJMPR RISK ASSESSMENTMRL的设定模型TMDI的计算是根据推荐的MRL值， 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

7、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

8、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 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。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 SPEGC/MS(SCAN

9、)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-FPDGC-ELCDDispersive-SPE Why use an SPE apparatus for “chemical filtration?” Dispersive-SPE involves the mixing of the sorbent with the extr

10、act in a mini-centrifuge tube to retain matrix interferants, but not analytes.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

11、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)ABCDABCDMethamidophos m/z 94Acephate m/z 136Omethoate m/z 156Carbofuran m/z 164Mevinphos m/z 127Fenvalerate m/z 167QUECHERS 方法参考论文

12、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 MethodThree PDP laboratories (California, Texas, and Washington) use MRM CDFA method： Fifty grams of homogenized samp

13、le is extracted withacetonitrile. Sodium chloride is added to the sample/acetonitrile mixture and agitated. All samples aresubjected to an initial C-18 solid phase extraction (SPE) clean-up step. The acetonitrile is evaporatedfrom one aliquot of each sample, which is reconstituted with acetone for o

14、rganophosphate analysis.Additional aliquots are cleaned up using florisil or aminopropyl SPE columns depending on the type ofcompound or the instrumental analysis to be conducted.Florida Department of Agriculture and Consumer Services Multi-Residue MethodThe Florida laboratory uses an acetonitrile-b

15、ased 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 chloride is added to the sample/acetonitril

16、e 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) analyses are cleaned up using an aminopropyl SP

17、E 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, strobilurinsModified Luke Multi-Residue MethodOne PDP laboratory (Oh

18、io) 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 final 10 mL extract is reconstituted in acetone

19、 and split for separate analysis using various instruments.New York Modified SPE MethodThe 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 some improvements based on the Luke extraction.50

20、g 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), and weak anion exchange (PSA). Compounds are eluted

21、 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 high performance liquid chromatography (HPLC)/tan

22、dem mass spectrometry (LC/MS/MS).NPDFPD/PFPDMSD / ITDGaschromatographic AnalysisECDFraction IECDFraction IIECDFraction IIIClean-up on silica gelGPC clean upDrying, Evaporation, FiltrationLiquid/liquid partition with NaCl and Cyclohexane/EthylacetateExtraction with Acetone/WaterPRINCIPLE OF THE PRINC

23、IPLE OF THE “S19S19”MULTI RESIDUE METHOD MULTI RESIDUE METHOD NPDFPD/PFPDMSD / ITDGC AnalysisECDFraction IECDFraction IIECDFraction IIICleanup on silica gelGPC cleanupDry, Evaporate, FilterLiquid/liquid partition with NaCl and Cyclohexane/EtAcExtraction with Acetone/WaterPRINCIPLE OF THE PRINCIPLE O

24、F THE “S19S19”MULTI RESIDUE METHOD MULTI RESIDUE METHOD Slide from Gnter Lach of EurofinsFruits and vegetablesExtraction: Module E 9 Sample weight: 50 gExtraction with ASEGPC with TACSDetection ECD and NPDGC column: DB 5, length 30 m, i. D. 0,25 mm, film thickness 0,25 m Relative frequency distribut

25、ion of chlorpyrifos residues in apple samples36Conclusions The typical sampling uncertainty For medium size crops with sample size 10CVS,med = 28% For small crops with sample size 10CVS,small CVS,medFor large crops with sample size 5CVS,large is about 40% (temporary estimate) 37SamplingConsideration

26、 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 are to be obtained. Preparation of the ana

27、lytical sampleThe 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 of analysis.For instance, to provide data for

28、 the estimation of maximum residue limits, the Codex Alimentarius Commission specifies the portion of commodities to which Maximum Residue Limits (MRLs) apply. Sample preparationAccording to the Codex Alimentarius, sample preparation is the procedure used, if required, to convert the laboratory samp

29、le into the analytical sample by removal of parts (soil, stones, bones) not to be analysed.For instance:for lettuce: remove dirty and withered leavesfor oranges: remove leaves and stemsThe method of sample preparation may be the source of substantial systematic and random errors, which cannot be est

30、imated.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. HomogenisationHomogenisation implies a mechanical size reduction

31、 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 degradation of pesticides. The homogenisation in the presenc

32、e 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 1% with 68% level of confidence取样系数与不确定度Apple sample

33、sConfidence intervals The uncertainty of the analytical result comprises:The expression can be modified to incorporate any additional stagesUncertainty components:The efficiency and uncertainty of sample processing can be tested utilising the “sampling constant”, KSStability of residues Systematic s

34、tudies 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, and even among varietiesRecent study on stability of residu

35、es during cryogenic processing : 94 of 106 pesticides tested were stablelosses of several pesticides -bitertanol (95%), heptenophos (50%), isophenphos (40%), and tolylfluanid (48%)- reported at Tamb did not occur during cryogenic processing Still needs to be demonstrated that has no detrimental effe

36、ct on some pesticidesExtraction 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 efficiency and method recovery. Filtration and concentration of the samp

37、le 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 of the analytical procedure can also be determined.Radiolabeled pe

38、sticides 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

39、确证手段GCMSLCMSMSMSSPR？ 快速、无损incorrectly identified or false positive resultsIn pesticide analysis, analytes in samples are often incorrectly identified or false positive results are reported. This may be due to:the presence of natural co-extractives or other contaminants, which mimic the characteristi

40、cs of some pesticides, or the lack of proper confirmation of the analyte identity.Retention time GLC analysisMass spectrometerHPLC and LC-MS/MS analysisDerivatisationTLC analysisDetermination criteria ELISADerivatisationEU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

41、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MS技术每种离子的识别点低分辨质谱1.0LR-MSn母离子1.0LR-MSn子离子1.5HRMS2.0HR-MSn母离子2.0HR-MSn子离子2.5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 不能超过下表相对丰度（%基峰）EI-GC-MS（相对）CI-GC-MS、GC-MSnLC-MS、LC-MSn（相对）50%10%20%20%到50%15%25%10

42、%到20%20%30%10%50%50%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 IR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

43、is it?QC proceduresSample handlingSamplingSample transportationSamples at laboratorySample storagePesticide standardsStock and working standardsExtractionContaminationInterferenceControl instrumental analysisDead volume peak shape testSolvent peak shape testSystem suitability tests (SSTs)Analytical

44、calibrationMatrix effectsAnalytical methods and recovery determinationsSpike or Recovery samplesDuplicate analysisRecovery determinationBlind samplesProficiency testingConfirmation of resultsReporting of resultsTools for IQCQC activitiesSample storageDuring the storage in the laboratory, the samples

45、 must be protected against physical, 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,polluti

46、on from the container. ExtractionAnalytical 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 t

47、o affect extraction efficiency 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 chromatographi

48、c system. The SST mixture 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. GC SST mixture for NP

49、D, FPD, PFPD and MSEPTCNeff, Sensitivity, Selectivity S to C (for PFPD) PropoxurInertness Tributylphosphate (TBP)Sensitivity, Selectivity P to CDimethoateAsymmetryPirimicarbSensitivity, Selectivity N to CChlorpyrifos methylResolution with parathion methylParathion methylResolution with chlorpyrifos

50、methylCarbarylInertnessChlorpyrifos ethylReference for RRTsQuinalphosResolution with methidathionMethidathionResolution with quinalphosHydrocarbon SelectivityPhosaloneNeff, RRTNPD SSTOptimized NPD SST NPD SST after injection of 40 sample extractChange of peak shape and RRT123Chlorpyrifos EthylAsym 1

51、.21 1.22 1.60123Dimethoate RRT Asym 0.690 1.56 0.691 1.82 0.708 3.33123EPTC RRT Asym 0.403 0.92 0.403 1.01 0.402 0.951- peak shape after the system maintenance;2 after injection of 20 sample extracts;3 after injection of 40 sample extractsVariation of P selectivity to carbon and MDQ of P in TBP duri

52、ng 3 year periodBead replaced, optimisedColumn cut, liner replacedCalibration: Matrix enhancement effectTools for IQCControl chartsControl charting is a suitable way of documenting IQC in analytical laboratories: it is used to monitor the stability of the measurement process and the analytical perfo

53、rmance over time. A control chart is a graphical plot of test results with respect to time or sequence of measurements, with established limits (upper and lower action/warning limits). A procedure is in statistical control when results consistently fall within the established control limits.Control

54、charts can be of different types:Charts for mean valuesCharts for individual valuesCharts for rangesInternal quality controlRe-analyse regularly analytical portions of positive samples. For 2 replicatesFor 3 replicatesSummary of most commonly used QC activitiesAnalysis of reagent blankAnalysis of blank samplesDuplicate analysisSystem suitabili