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Developing a Pesticide Analysis Program to Meet International Food Trade Requirements: The Codex Alimentarius Influence Philip L. Wylie, Ph.D. Agilent.

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Presentation on theme: "Developing a Pesticide Analysis Program to Meet International Food Trade Requirements: The Codex Alimentarius Influence Philip L. Wylie, Ph.D. Agilent."— Presentation transcript:

1 Developing a Pesticide Analysis Program to Meet International Food Trade Requirements: The Codex Alimentarius Influence Philip L. Wylie, Ph.D. Agilent Technologies Wilmington, DE USA This talk is intended to introduce the audience to Codex Alimentarius and to help them to choose the right instrumentation for pesticide analysis in fruits and vegetables.

2 Presentation Outline Pesticide use around the world
Pesticides and International Food Trade Codex Alimentarius Starting a Pesticide Monitoring Program for international food trade Where to get help & find requirements Your instrumentation investment - what equipment is needed

3 Pesticides Usage More than 700 pesticides are registered for use WW
About 2.2 billion kg of pesticide used each year WW 1995 WW pesticide sales = $29 billion Some very toxic pesticides are banned in many countries but may still be used in others: Endrin, DDT, lindane, aldrin, chlordane, and many others No standardization of Maximum Residue Limits (MRLs) in food Banned or highly restricted pesticides have been “dumped” in developing countries Modern agriculture has become dependent upon pesticides to grow enough food for the world’s increasing population. The term “pesticide” generally refers to any biological or chemical agent used for crop protection or pest control. They can be applied to the soil, the plant, the harvested crop, or even shipping containers to kill pests such as weeds, insects, nematodes, fungi, or other organisms that might decrease crop yields or cause crops to spoil prematurely. Although more than 700 different pesticides are registered for use world wide, most laboratories only analyze fruits and vegetables for a fraction of them. Many of the first pesticides to be developed have since been found to be toxic to organisms (including humans) that were not the intended targets. Many of those (such as DDT) have been banned in most countries, but are still used by a few. In some cases, excess stock of banned pesticides have been sold to developing countries that may not have such strict regulations. This is known as “dumping” of the pesticides. Many of the “persistent organic pollutants” that have been the subject of international meetings and treaties are pesticide compounds.

4 Pesticide MRL Influence on International Food Trade
Many countries have rigorous pesticide registration processes Many countries have created their own MRLs for pesticides based on local laws Within one country MRLs are often different on different food types (pear MRL may not be the same as onion MRL) MRLs are often different in different countries Differences can lead to Intentional or unintentional trade barriers Many of the highly developed countries in the world have strict regulations for the registration of new pesticides. A pesticide may not be used on any crop unless it is specifically registered for use on that crop. Regulators take into consideration many factors such as the need for the pesticide; availability of safer alternatives; the toxicity of the pesticide in humans, birds, fish, and other wildlife; its persistence in the environment, its likelihood of causing cancer, etc. For a single pesticide, the Maximum Residue Limit (MRL) at the time of harvest may be different for different crops. For example, the MRL could be 0.1 ppm on apples and 5 ppm on whole oranges. A pesticide could be registered for use only on cotton and, therefore, must not be used on any other crop in that country. Because individual countries have the power to regulate pesticide use within their borders, many countries have their own agencies for pesticide registration. Consequently, many countries have set different MRLs for the same pesticide on the same crop (see the next slide). Because of these differences, foods that contain acceptable levels of pesticide residues in one country could be rejected in another that has a lower MRL.

5 Comparison of Codex MRLs for Apples (ppm) to Some National MRLs
This is one example of differences in MRL values among different countries for the same pesticide on the same commodity. In this case we are looking at MRL values for six different pesticides that are used on apples. For example, the US allows 25 ppm of Captan residues on apples while Japan allows only 5 ppm and France only 3 ppm. Where there blanks (such as with Dimethoate in Japan) it means that this pesticide is not registered for use on apples in that country. Taiwan has adopted some MRL values from the US and Codex Alimentarius. We will discuss the Codex Alimentarius organization and its attempts to “harmonize” pesticide residue levels a little later in this talk. These variations in MRL values in different countries can lead to significant barriers to food trade around the world. Food that’s acceptable in one country may not be allowed into another because of its pesticide residue regulations. * In some cases Taiwan uses Codex or US MRLs Reference: Northwest Horticultural Council Export Manual Available at:

6 Pesticide Related Trade Barriers
Pesticides used legally on a crop in an exporting country are not registered for use in the importing country - e.g. banana growers may use a safe pesticide but an importing country doesn’t need or use that pesticide There are different ways that pesticide use and residue regulations can lead to trade barriers between countries. In this example, a pesticide may be used on bananas that are grown in a tropical country. That country has determined that this pesticide is safe for use on bananas. However, this pesticide might not be used at all in a northern country where bananas are not grown and it may not have an MRL value for that pesticide on bananas. The importing country could decide to reject these bananas because of its pesticide residues.

7 Pesticide Related Trade Barriers
As seen for apples, MRLs for a given pesticide can vary widely from one country to the next. This too can create trade barriers. A crop that is acceptable in the country where it is grown (residues below that countries MRL values) might not be imported into another country where the MRLs are set lower. Another problem can arise when a crop is found to have unacceptably high residues in the country where it is grown. In this case, the grower or packer might ship it to another country where the MRL is higher or, perhaps, to a developing country that does not have a laboratory to measure pesticide residue levels. This could put the people in the developing country at risk. Different MRLs - Residues at legal levels in exporting country are too high for the importing country

8 Pesticide Related Trade Barriers
Past DDT Spraying Contamination from “extraneous” pesticides such as DDT that may be in the soil from previous use Many pesticides that were once widely used are now banned in most countries because they were found to be toxic to wildlife or humans. The most famous of these is DDT which was used widely to kill insects (including mosquitoes) in much of the world. Among other problems, it bio-accumulated in birds causing a thinning of their egg shells and a reduction of many bird populations. DDT is still used in some malaria-plagued countries where there is no cheap alternative for mosquito control. DDT (or its metabolites/decomposition products - DDE and DDD) may contaminate the soil from past use and can still show up in fruits and vegetables grown in that soil. This could lead to rejection of the commodity by an importing country.

9 Economic Trade Barriers
Designed to protect farm products of the importing country May be disguised as health or safety measures May be a response to consumer misconceptions about the dangers Pesticide or drug MRLs could be set so low that it prevents imports Economic trade barriers are always controversial, but they are still very common. A country may use pesticide (or drug) residue levels in vegetable (or animal) products as an excuse to reject certain imports, thereby protecting their own farmers from outside competition. In some cases consumers actually promote trade barriers by lobbying their government against the importation of food containing certain pesticides, drugs, or other additives. This can lead regulators in that country to ban a pesticide or to set its MRL at an extremely low level, essentially banning imports from any country where that pesticide is used. Although this is not the subject of this talk, there is a great deal of resistance in some parts of the world to the importation of genetically modified organisms (GMOs). Fruit spoils when rejected by importer

10 United Nations Creates the Codex Alimentarius Commission
“Codex Alimentarius” is Latin for “Food Code” Created in by the United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) Now has 165 Member Countries representing 97% of the world’s population In the early 1960s, the United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) realized that lack of uniformity in food regulations was preventing world trade. Together, the FAO and WHO created the Codex Alimentarius Commission in an attempt to rectify this problem. “Codex Alimentarius” is Latin for Food Code or Food Law. Most countries in the world are members of the Codex Commission.

11 Codex Alimentarius Commission Goals
To develop a compilation of scientifically-based standards, codes of practice and guidelines for the production, processing and shipping of food... In order “to protect the health of consumers and to ensure fair practices in the food trade” To “harmonize” food safety standards world wide To assist developing countries in meeting these standards One of the main goals of the Codex Commission is to “harmonize” food laws around the world. That is, they hope to create a set of standardized food regulations that all countries would recognize and adopt. This would bring down trade barriers of the type discussed in the previous slides. Special subcommittees composed of experts in the field would make recommendations for regulations that would be adopted by the member nations. Two of these groups are known as the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA). These are discussed in the next slide. Because most developing countries did not have laboratories capable of monitoring pesticide usage, pesticide quality, and residue levels, they were at a big disadvantage. Many countries still do not have this capability and are unable to protect the health of their own citizens, to prevent pesticide dumping, and to promote acceptance of their food exports. Therefore, one of the goals of the Codex Commission is to assist developing countries by providing the information and training required to set up monitoring labs and to perform the necessary analyses.

12 Organizations Associated with the Codex Alimentarius
Joint FAO/WHO Meeting on Pesticide Residues (JMPR) JMPR is independent of the CODEX Commission JMPR recommends maximum residue limits (MRL) Joint FAO/WHO Expert Committee on Food Additives (JECFA) JECFA is independent of the CODEX Commission JECFA recommends limits for veterinary drug residues, food additives and other contaminants International Atomic Energy Agency (IAEA) FAO/IAEA Training and Reference Centre for Food and Pesticide Control ( The JMPR is a committee of scientific experts who come from the member countries. They meet periodically to consider new pesticide MRLs and to make recommendations to the Codex Commission. Their goal is to use available scientific data to set MRLs that will protect people and the environment without creating artificial trade barriers. This can be a difficult process because scientists don’t always agree on data interpretation or on the significance of experimental results. The JECFA serves a similar function as the JMPR, except its focus is on drug residues in animal products and other food contaminants. It’s not obvious why the International Atomic Energy Agency would be involved in developing food regulations, but they participate in training scientists. The FAO and IAEA have a training center in Austria. They organize various courses to train technicians and scientists from the developing world. Courses may last several weeks and may be given at the FAO/IAEA Training Center or at another laboratory elsewhere in the world. Trainees must be nominated by their government to participate.

13 Codex Alimentarius: Has developed
- Food standards for 237 different commodities - 41 codes of hygienic or technological practice - 25 guidelines for contaminants MRLs for pesticide residues Has evaluated food additives - 54 veterinary drugs - 185 different pesticides The member nations of Codex have adopted a large number of standards for food, food additives, and chemical residues (pesticide and drug). Of the approximately 700 pesticides in use today, Codex has evaluated This may seem like slow progress, but this list includes many of the widely used pesticides so it is more comprehensive than you might think. A single MRL is the residue limit for a single pesticide on a single commodity. For example, Captan has maximum residue limits of 20 ppm for strawberries and blueberries, 15 ppm for peaches and tomatoes, and 25 ppm for apples and pears. So, Codex has set individual MRLs for captan.

14 Contents of the Codex Alimentarius
Volume 1A - General requirements Volume 1B - General requirements (food hygiene) Volume 2A - Pesticide residues in foods (general texts) Volume 2B - Pesticide residues in foods (maximum residue limits) Volume 3 - Residues of veterinary drugs in foods Volume 4 - Foods for special dietary uses (including foods for infants and children) Volume 5A - Processed and quick-frozen fruits and vegetables Codex has produced 13 volumes of food standards (counting parts A and B as a single volume), of which one (Volume 2A and 2B) deals with pesticide residues in food. All 13 of the volumes are available in English, Spanish, and French.

15 Contents of the Codex Alimentarius
Volume 5B - Fresh fruits and vegetables Volume 6 - Fruit juices Volume 7 - Cereals, pulses (legumes) and derived products and vegetable proteins Volume 8 - Fats and oils and related products Volume 9 - Fish and fishery products Volume 10 - Meat and meat products; soups and broths Volume 11 - Sugars, cocoa products and chocolate and miscellaneous products Volume 12 - Milk and milk products Volume 13 - Methods of analysis and sampling Codex Alimentarius offers standards for much more than pesticide and drug resicues in food. There are regulations for storage, packaging, composition, food additives, etc.

16 Contents of Vol. 2A: “Pesticide Residues in Food - Methods of Analysis and Sampling”
Recommended Methods of Sampling for the Determination of Pesticide Residues for Compliance with MRLs Recommended methods of analysis for pesticide residues Portion of commodities to which codex MRLs apply and which is analyzed Guidelines on Good Laboratory Practice in pesticide residue analysis Suggested analytical methods Volume 2A gives information such as: 1. How to choose, collect, transport, preserve, and prepare samples for analysis 2. The portion of the commodity that should be analyzed (For example, whole cantaloupe melons and whole oranges should be analyzed including the skin. However, apricots, cherries, peaches and other stone fruits are analyzed after removing the stone (seed) portion. 3. Some basic information on good laboratory practices - there will be more discussion of this later in the talk. 4. Suggested analytical methods. Many different references are given for pesticide residue methods. This chapter can be very confusing and many, if not most, of the methods listed are not widely used. Many of these are out of date. I have given some modern, up-to-date methods later in the talk. I recommend that customers first look at these multi-residue methods and use most of the methods listed in Codex only if one of the other methods cannot be used.

17 Where to get the Codex Alimentarius
Sales and Marketing Group Food and Agriculture Organization of the United Nations Viale delle Terme di Caracalla Rome, Italy Some parts of the Codex Alimentarius can be viewed on the web at: Available in English, French, & Spanish Copies of the Codex Alimentarius can be ordered from the address shown. One can purchase an individual volume or the entire set. Recently, the Codex volumes could be downloaded from the web, but the web site has changed and now I can’t find them anymore. However, you can still find pesticide and drug MRLs on the web by starting at the Codex home page (address shown).

18 Imagine the Following Scenario in Your Country: Sunshine Island
Sunshine Island grows a variety of tropical fruits and vegetables Exports are growing, which benefits the local economy But, sometimes shipments are rejected by the importer because of pesticide residues - it has become a costly problem Sunshine Island has not monitored pesticide residues in the past You must set up a laboratory to analyze your food to meet Codex Pesticide MRLs This is the situation faced by many countries in the developing world today. Food trade has increased enormously in recent decades. Many developing countries are looking for world wide markets for their food products. However, they may not have the laboratory facilities to test their exports and imports for pesticide and drug residues. It is becoming important for these countries to set up laboratories to run these analyses. We will use the example of “Sunshine Island” to illustrate some of the questions that must be answered in the process of setting up a new pesticide residue lab. There are many references given that will lead interested people to resources that can help them choose methods and validate them in their own labs. Of course, Agilent can provide the hardware and a lot of experience to help these labs get started.

19 You have Some Important Questions
What are the MRLs for pesticides in our export products? What extraction procedures should we use? What analytical techniques do we need? What instrumentation do we need? How do we validate our methods? Where can we get help in developing Standard Operating Procedures (SOPs)? Where can we get training? These are some of the important questions that must be asked when setting up a new laboratory for pesticide residue analysis. Will you be analyzing only products for export, or will you be analyzing imports and food sold locally. Most regulatory labs do all three, so they need to analyze for many different pesticides in a wide variety of food products. This means that they need to use multi-residue methods that have been validated for many kinds of produce. We will attempt to answer the questions that are listed in this slide. We do not have time in this talk to give the exact details, but many references are listed so that the audience can gather information from the original sources.

20 Searching the Codex Database to Find all Pesticide MRLs for Papayas
As we saw earlier, many countries have developed their own MRLs, which can be different from one country to the next. However, when international trade disputes arise, the World Trade Organization recognizes Codex MRLs. The Codex MRLs are listed in Volume 2B of the Codex Alimentarius and they are available on the web. Using the web, it is easy to search the Codex database in one of two ways: 1) You can choose a fruit or vegetable and find all pesticides that have a Codex MRL for that commodity. 2) You can choose a pesticide and list all of the fruits and vegetables on which it can be used. In this case, we have highlighted PAPAYA and will search for all pesticides that have an MRL for that fruit. Also available in Volume 2B of the Codex Alimentarius

21 Chinomethionat, Dithiocarbamates, and Prochloraz have Codex MRLs for Papayas
Pesticide MRL Value (ppm) We find MRL values for Chinomethionat, Dithiocarbamates (a class of pesticides), and Prochloraz. MRL values are usually expressed in ppm (mg/kg).

22 Best Approach for Choosing Extraction and Analysis Methods
Choose a method already in use by experienced pesticide analysts It will already be validated in at least one lab Make minor adaptations as needed for: differences in commodities differences in analytical equipment Validate the method in your laboratory There is usually no need to develop new extraction and analysis methods because many useful procedures are already published. However, laboratories do occasionally make improvements. Some of the methods suggested in the Codex Alimentarius Volume 2A and very old and outdated, although they probably work. The best choice is to use a published method that has been validated and is in wide use. This will save a LOT of time and trouble. Labs may need to make minor changes in procedures to accommodate their personnel, laboratory equipment, and instrumentation. The only way to be sure that analyses are being performed correctly is to validate the methods in your own lab. Once the method is validated, it is a good idea to run performance check samples on a regular basis to be sure that your methods are still providing good results. Performance check samples are often prepared and distributed by an organization set up for that purpose. The laboratory should analyze the samples “blind” and should compare their results with the known values after the analysis is complete.

23 Where can we find Good Validated Methods?
Florida Department of Agriculture and Consumer services J. Cook, M.P. Beckett, B. Reliford, W. Hammock, M. Engel (1999) J. AOAC Int. 82, California Department of Food and Agriculture ( Multiresidue Screen for Pesticides in Fruits and Vegetables (1995) California Department of Food and Agriculture, Sacramento, CA, USA summary 1-2 S.M. Lee, M.L. Papathakis, H.M.C. Feng, G.C. Hunter, J.E. Carr (1991) Fresenius J. Anal. Chem. 339, In the United States, two of the most active pesticide residue laboratories are the Florida Department of Agriculture and Consumer Services and the California Department of Food and Agriculture. California and Florida are important agricultural states and have been analyzing pesticide residues in food for many, many years. They use very similar extraction procedures. A recent publication by Cook, Beckett, Reliford, Hammock, and Engel gives all the details of the Florida extraction (and analysis) procedure. This is an excellent article and should be read by anyone wishing to analyze pesticide residues in food. The labs in Florida and California use mostly Agilent equipment.

24 Where can we find Good Validated Methods?
Ministry of Public Health, Welfare and Sport, The Netherlands Analytical Methods for Pesticide Residues in Foodstuffs, 6th ed. (1996) General Inspectorate for Health Protection Ministry of Public Health, Welfare and Sport (The Netherlands) Pesticide Analytical Manual (PAM) U. S. Food and Drug Administration Center for Food Safety and Applied Nutrition Office of Plant and Dairy Foods and Beverages ; Updated October, 1999 Can download from the WWW at: Includes a lot of basic information on chromatography The Ministry of Public Health Welfare and Sport in The Netherlands has published a very comprehensive manual on pesticide analysis. This laboratory likes to use ion traps (from Varian) for some screening and for confirmation, although they do not run it in the MS/MS mode. Ion traps have problems with some of the pesticides and also have major problems with matrix. If one of your customers in interested in GC/ion trap MS, please contact Jim Yano (Agilent’s MSD product manager). He can explain why GC/ ion traps are a poor choice for pesticide residue analysis. The United States Food and Drug Administration has developed many popular extraction and analysis methods which are published in the Pesticide Analytical Manual (PAM). The manual gives a great deal of basic information about individual pesticides, extraction methods, and analytical techniques. Unfortunately, some of their techniques are rather outdated. Still, the PAM is a very valuable resource and each pesticide residue lab should have a copy. The PAM also has methods for the analysis of single pesticide residues when these pesticides cannot be determined by a multi-residue method.

25 A Typical Fruit & Vegetable Extraction Procedure
This procedure is used by the Florida Department of Agriculture and Consumer Services as described in the following journal article: J. Cook, M.P. Beckett, B. Reliford, W. Hammock, M. Engel (1999) J. AOAC Int. 82, We will outline the extraction procedure used routinely by the Florida Department of Agriculture and Consumer Services (which is very similar to the one used in California). This will help us to understand the importance of sample cleanup and why selective detectors are so important. (The alligator can be found in any of Florida’s fresh water swamps, streams, and lakes. It can grow to 4 meters or more in length.)

26 Florida Department of Food and Agriculture Extraction Steps
Weigh 50 g Homogenized Fruit/Vegetable Sample into 250-mL Polyethylene (PE) Bottle Add spiking solution and 100 mL CH3CN, cap & shake 3 min Acetonitrile Extract Gravity filtration Filtered Acetonitrile Extract C-18 SPE under N2 pressure; Collect in 250 mL PE bottle containing 10 g NaCl Normally, several kilograms of the food sample are homogenized in a large blender. Most labs use industrial blenders from Robot Coupe ( By blending a large sample, the 50-g sample that is extracted is more representative. California and Florida do the initial extraction using acetonitrile solvent. The PAM method uses acetone. Food extracts can be very dirty, so in recent years, solid phase extraction (SPE) has become widely used for sample cleanup. Extract & NaCl in Bottle Shake 2 min; Centrifuge rpm; Decant into 70-mL SPE reservoir; Drain off H2O layer Acetonitrile Extract Separated from H2O

27 Florida Department of Food and Agriculture Extraction Steps
Acetonitrile Extract Separated from H2O Collect 15 mL in 100-mL Beaker Collect 15 mL in 100-mL Beaker P Fraction C Fraction Aminopropyl SPE on 4 mL of CH3CN extract; Elute with 3% methanol in acetone Aminopropyl SPE-Cleaned Extract Evaporate using N2 just to dryness; Add 1 mL MeOH; Filter into 2-mL ALS vial The initial acetonitrile extract is divided into three aliquots - one will eventually be analyzed by HPLC for carbamate pesticides, one (the “P” fraction) will undergo further cleanup and will be analyzed for organophosphorus pesticides, and the last aliquot (the “C” fraction) will be analyzed for organochlorine residues. In the past, carbamates were almost always analyzed by HPLC with post column derivatization and fluorescence detection. Such a system is sold by Pickering Laboratories ( In the last few years, many labs have switched to GC/MS for carbamate analysis. Though more expensive, MS gives some level of confirmation and it is very sensitive. Final Extract for Carbamate Analysis HPLC/Fluorescence or HPLC/MS

28 Florida Department of Food and Agriculture Extraction Steps
15 mL CH3CN Extract (P Fraction) Evaporate to ~2 mL on steam bath; Air dry in hood Dry Extract Add 2 mL Acetone; Swirl to dissolve Acetone Solution Pour into 16 X 125 mm screw cap test tube; refrigerate >15 min to precipitate NaCl Final Extract (P Fraction) It would not be a good idea to inject an acetonitrile extract into a GC/NPD (for phosphorus and nitrogen detection) so the acetonitrile is removed and acetone is added. The solution is cooled to precipitate NaCl and the solution is ready for analysis. This solution is usually analyzed by GC with NPD and FPD detectors. GC Analysis (N, P, S Selective Detectors or GC/MS)

29 Florida Department of Food and Agriculture Extraction Steps
15 mL CH3CN Extract (C Fraction) Evaporate to ~2 mL on steam bath; Air dry in hood Dry Extract Add 2 mL 85:15 hexane:acetone; Swirl to dissolve Hexane:Acetone Solution Florisil SPE; Rinse with 85:15 hexane:acetone; Collect in 15-mL graduated test tube Florisil-Cleaned C Fraction in Hexane/Acetone The third aliquot of the acetonitrile extract is cleaned up using a florisil solid phase extraction tube. This fraction is analyzed by GC/ECD or by GC/XSD. The ECD is extremely sensitive for polychlorinated pesticides and PCBs, but it is not completely selective for halogens. That is why it is important use the florisil clean-up step. The XSD is a detector sold by OI Analytical, one of Agilent’s channel partners. It responds to halogens and is used by some labs for pesticide analysis. Reduce volume under N2; Adjust to 4 mL with isooctane GC Analysis (Halogen-Selective Detector or GC/MS)

30 Almost All Pesticides Contain Heteroatoms (Oxygen is too common)
Element-selective GC detectors are commonly used for pesticide analysis P N H O S C 3 Acephate Cl Atrazine + - F Trifluralin Heptachlor Element selective detectors have been widely used for pesticide analysis because most pesticides contain heteroatoms. Cl, P, N, and S are the most common heteroatoms found but some pesticides also contain F, Br, and metals. Of course, O is found in the majority of pesticides but it is so widely distributed in natural products, it would be useless to identify pesticides using oxygen-selective detection.

31 GC Detectors for Pesticide Analysis
This table lists the most commonly used detectors for the GC analysis of pesticides. Of these, the NPD (for nitrogen and phosphorus), FPD (for phosphorus or sulfur), and the ECD (for Br, Cl, and F) are the most widely used. The Electrolytic Conductivity Detector (ELCD) responds to halogens, but it is VERY difficult to use and maintain. Most chemists would rather have a tooth pulled than have to run an ELCD. The Halogen-Specific Detector is newer and seems to be easier to operate and maintain. Both the ELCD and XSD are sold by OI Corporation, which is an Agilent channel partner. For most labs it would be best to have NPD, FPD, and ECD detectors for the initial analysis of samples and an MSD for confirmation. Another excellent detector for pesticide analysis is the Atomic Emission Detector (AED). However, Agilent recently sold this business to JAS (a channel partner) and it is probably too expensive for most labs that are just beginning to analyze pesticide residues in food.

32 GC Detectors for Pesticide Analysis
It is probably safe to say that all pesticide laboratories in the US and Western European countries have at least one GC/MS system. In most cases, it is used for confirmation of pesticide identities after they have been detected by element-selective detectors. The Agilent 6890/5973 system has an excellent reputation in these laboratories because of its sensitivity, ease of use, quality ChemStation software, and ability to give good results even when there is a high background of co-extracted plant material. As seen later in this talk, chemists from Canada have developed a pesticide screening method that uses only the GC/MSD. This method is routinely used to screen fruits and vegetables in Canadian government laboratories. However, this is a SIM method and some pesticides are only identified by their retention time and two ions (the target ion and one qualifier). This may not always be enough evidence for export to many other countries.

33 Analytical Techniques and Instrumentation for Pesticide Analysis
Basic target pesticide analysis (entry solution) Dual column GC(s) with selective detectors ECD for organochlorine pesticides NPD for organonitrogen pesticides and organophosphorus pesticides FPD for organophosphorus pesticides 30-m X 0.25-mm X 0.25 um HP-5MS column (P/N 19091S-433) & a more polar column for confirmation LC with post-column derivitization and fluorescence detection (Pickering System) Analysis of carbamate pesticides In the next few slides, we will suggest hardware solutions for labs that need to analyze pesticide residues in food. The exact configurations will depend somewhat on the pesticides and foods to be analyzed. Assuming that the laboratory needs to screen for organochlorine, organophosphorus, and organonitrogen pesticides, it will be necessary to have halogen-, phosphorus-, and nitrogen-selective detectors. Therefore, we recommend using the u-ECD, FPD, and NPD. Because food extracts always contain other co-extracted materials (even after SPE), peaks seen in one of these selective detectors must be confirmed using some other analysis. The easiest method is to repeat the analysis using a column with a more polar phase. Peaks that match the retention time of a pesticide on both columns is considered to be identified correctly. Often the analysis on the second column can be performed in the same run by splitting the injection between two columns, each of which is connected to a separate detector. Of course, the best choice for dual column confirmation is to have a 6890 configured with two split/splitless inlets and two identical detectors (ECD, NPD, or FPD). The first column used should be an HP-5MS. We recommend the 30-m X 0.25-mm X 0.25-um HP-5MS, but some labs might want to use a megabore column because it is more “forgiving” when injecting dirty samples.

34 Analytical Techniques and Instrumentation for Pesticide Analysis
Typical Capability of Laboratories in the US and Europe GC(s) with selective detectors ECD for organochlorine pesticides NPD for organonitrogen pesticides and organophosphorus pesticides FPD for organophosphorus pesticides GC/MSD for pesticide confirmation LC/MSD for carbamates & other non-volatile or sensitive pesticides Most pesticide regulatory labs in the US and Europe use selective detectors for initial pesticide screening and GC/MS for pesticide confirmation. This is a powerful combination of complimentary tools. These analytical results will stand up in court in cases where a violation has been found. In the last several years, most of these laboratories have purchased LC/MS equipment for the analysis of carbamates and other pesticides that cannot be analyzed by GC. Of LC/MS detectors offered by Agilent, the quadrupole MSD is the easiest to use for routine analysis. The ion trap LC/MS system is better for research and method development. It should be noted here that many laboratories actually prefer LC/MS with a triple quad detector because of its added selectivity and sensitivity. Of course, Agilent does not sell a triple quad for GC or LC, but we have announced our intention to introduce a Q-TOF instrument within the next two years, which may be an excellent tool for pesticide analysis. However, WE CANNOT SELL THIS INSTRUMENT NOW. For those labs that are just beginning with LC/MS, it is important to emphasize the ease of use and ruggedness of our quadrupole LC/MS system. The software is built on familiar Agilent ChemStation platforms so it will be easy for operators to learn.

35 Advanced Pesticide Analysis
Use Retention Time Locking (RTL) for GC & GC/MS 30-m X 0.25-mm X 0.25 um HP-5MS column (P/N 19091S-433) locked to Agilent pesticide method Benefits of RTL Use Agilent GC and GC/MS RTL Libraries for pesticide identification Lock all GC and GC/MS methods All GC & GC/MS analyses have same RT All runs match the RTL Library RTs Easier pesticide identification Easier pesticide confirmations by GC/MS Agilent provides the most comprehensive set of pesticide analysis tools available in the world. Not only do we have the hardware required for pesticide analysis, we invented retention time locking (RTL) and retention time locked database searching for GC and GC/MS. Agilent offers two databases containing the locked retention times for 567 pesticides and suspected endocrine disrupters - one for GC and one for GC/MS. Agilent’s RTL software for GC and GC/MS is a valuable tool for identifying and confirming known and unknown pesticides. RTL allows the lab to match GC and GC/MS retention times which is extremely helpful when comparing chromatograms. The point of this talk is not to emphasize RTL solutions for pesticide analysis, but that would be an excellent talk to combine with this Codex presentation in a longer seminar.

36 Is a GC/MSD Really More Expensive than GCs with Selective Detectors?
Dual Column GC/NPD Dual Column GC/FPD Dual Column GC/ECD It will depend on your samples, the pesticides you analyze, and what level of confirmation is required. As shown in the following slide, Canadian chemists have developed a pesticide screening method for the volatile pesticides that only uses GC/MS. This is a SIM method that requires 2 injections to screen for more than 200 pesticides. Depending on the laboratory needs, it might be cheaper to implement this approach on a single GC/MS system than to purchase 3 GC systems configured for dual column confirmation with selective detectors. The Canadian screening method often relies on only the target ion and one qualifier to identify the pesticide so it is not acceptable to all regulating bodies. Before choosing this approach as the only screening method, it would be important for the laboratory to check with all regulating authorities (domestic and foreign) to be sure that they will accept these results. or GC/MSD

37 Canada Analyzes Pesticides using only GC/MS and LC/Fluorescence
Julie Fillion, François Sauvé, and Jennifer Selwyn, Multiresidue Method for the Determination of Residues of 251 Pesticides in Fruits and Vegetables by Gas Chromatography/Mass Spectrometry and Liquid Chromatography with Fluorescence Detection, J. AOAC Int., 83 (3), and J. AOAC Int., 78 (5), As mentioned in the last slide, Canada has developed a screening method for more than 200 pesticides. There are two good journal references to this method which give important details that would allow one to repeat this analysis. Note the precaution discussed in the previous slide. The level of confirmation for some pesticides is not adequate for all regulating authorities. LC with the Pickering system or LC/MS is still required for the carbamates. Note: Some countries do not “recognize” this method because many pesticides are identified by only two ions

38 Help in Validating Your Methods
Start with a method that has already been validated Use the following resource prepared by the Joint FAO/IAEA Expert Consultation on Method Validation Guidelines for single-laboratory Validation of Analytical Methods for Trace-level Concentrations of Organic Chemicals Available on the www at: Method validation can be tedious and could take several weeks to complete in some cases. It is very helpful to begin with a validated method that is widely accepted elsewhere in the world. The Joint Food and Agriculture Organization/International Atomic Energy Association (FAO/IAEA) expert Consultation on Method Validation has published a very useful set of guidelines for validating a method in a single laboratory. It is usually best to validate an analytical method in several laboratories, but this is usually not practical. It’s much easier to begin with a validated method and validate it in your own lab by following the guidelines mentioned above.

39 Help in Developing Standard Operating Procedures (SOPs)?
The Pesticide Data Program of the US Department of Agriculture has published all of their SOPs on the www at: SOPs for Administration of sampling & lab processes sampling procedures laboratory analysis standards, laboratory operations, quality control, and data handling Includes 60 SOPs Standard operating procedures are like a detailed recipe for preparing food. It gives all of the steps, measurements, etc. required to perform the task. These are usually broken down into small tasks, such as preparation of a standard or installing a GC column. In a pesticide laboratory, SOPs are usually available for every procedure from collecting the samples, analyzing them, and archiving the data. A very good set of SOPs has been developed by the Pesticide Data Program (PDP) which is run by the US Department of Agriculture. This is a well-run program that uses state of the art techniques to measure pesticide residues at the lowest possible levels in fruits and vegetables. Fortunately, the PDP program has published their SOPs on the web and anyone can download them. Of course, modifications would normally be required, but these are good examples of what is required.

40 Training for Pesticide Analysis
Courses are offered by the UN Food and Agriculture Organization with The International Atomic Energy Agency Courses are offered on: pesticide residue analysis, mycotoxin analysis, & quality control of pesticide products Requires nomination by your government See: The Natural Resources Institute offers 13-week Pesticide Residue Analysis Training & related Courses at the University of Greenwich See: Agilent Technologies Ask your Agilent representative Universities all over the world offer degrees in chemistry, environmental toxicology, and similar fields. It is good, of course, to have this background but we know that not everyone in a pesticide laboratory will have education in that field. Knowing this, the United Nations has developed courses in several areas of food safety analysis that are given jointly by the FAO and IAEA. To attend, you must be nominated by your government. Some courses are several weeks long. Agilent offers many courses on general instrument operation that would be extremely useful for anyone who is new to modern GC, GC/MS, LC, or LC/MS. At this point, we do not offer courses specifically for pesticide residue analysis.

41 Conclusions International food trade requires pesticide residue analysis Codex Alimentarius provides harmonized regulations for foods, including pesticide MRLs Agilent Technologies provides all of the required analytical instruments GC, GC/MS, LC, LC/MS, LC/MS/MS, ICP/MS, Columns, Supplies analytical methods based on Retention Time Locking RTL pesticide databases for GC and GC/MS expert consultation and training The Codex Alimentarius Commission is attempting to increase world food trade and the participation by developing countries by developing a universal set of food standards and regulations. The process began about 40 years ago and continues today. All countries need to have pesticide analysis laboratories in order to protect the health of their own people and to facilitate export of their domestic foods. Although it may be expensive to set up and maintain such a laboratory, there can be a large return on the investment. Pesticide analysis can ensure the safety of domestically produced food and make it acceptable to importing countries. Agilent equipment and methods are used world wide for pesticide residue analysis. We have the experience and the reliable products that can make any pesticide laboratory successful.

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