1. Dedi FardiazWHO SEARO, Jakarta, 0612071 Key Issues in Analysis of Pesticides and Heavy Metals Dedi Fardiaz National Agency for Drug and Food Control Republic of Indonesia WHO SEARO Regional Workshop on Total Diet Studies Jakarta, Indonesia, 5-7 December 2007 Key Issues in Analysis of Pesticides and Heavy Metals Dedi Fardiaz National Agency for Drug and Food Control Republic of Indonesia WHO SEARO Regional Workshop on Total Diet Studies Jakarta, Indonesia, 5-7 December 2007

2. Dedi FardiazWHO SEARO, Jakarta, 0612072 Terms for Mass Fraction Ranges Term Mass Fraction (%) Mass Fraction Major 0.1 - 100 1 - 1000 g/kg Minor 0.001 - 0.1 10 - 1000 mg/kg Trace0.000001 - 0.001 0.01 - 10 mg/kg Ultra Trace < 0.000001 < 10  g/kg Pesticide Residues in Food? Heavy Metals in Food? Attention to detail analytical procedure is essential Terms for Mass Fraction Ranges Term Mass Fraction (%) Mass Fraction Major 0.1 - 100 1 - 1000 g/kg Minor 0.001 - 0.1 10 - 1000 mg/kg Trace0.000001 - 0.001 0.01 - 10 mg/kg Ultra Trace < 0.000001 < 10  g/kg Pesticide Residues in Food? Heavy Metals in Food? Attention to detail analytical procedure is essential

3. Dedi FardiazWHO SEARO, Jakarta, 0612073 X  (“True Value”) Testing Target Accuracy Precision accuracy precision accuracy & precision

4. Dedi FardiazWHO SEARO, Jakarta, 0612074 X X  (“True Value”) Testing Target Accuracy Precision Reduce the Uncertainty by reducing the following errors: - Measurement Error - Sampling Error - Calibration Error

5. Dedi FardiazWHO SEARO, Jakarta, 0612075 What instrument do we use? Gas Chromatograph (GC) High Performance Liquid Chromatograph (HPLC) Gas Chromatograph/Mass Spectrometer (GC/MS) Liquid Chromatograph/Mass Spectrometer (LC/MS) GC/MS-MS LC/MS-MS Atomic Absorption Spectrophotometer (AAS) Electrothermal Atomization (graphite furnace) AAS Others? What is important? The instrument or the analytical process What instrument do we use? Gas Chromatograph (GC) High Performance Liquid Chromatograph (HPLC) Gas Chromatograph/Mass Spectrometer (GC/MS) Liquid Chromatograph/Mass Spectrometer (LC/MS) GC/MS-MS LC/MS-MS Atomic Absorption Spectrophotometer (AAS) Electrothermal Atomization (graphite furnace) AAS Others? What is important? The instrument or the analytical process

6. Dedi FardiazWHO SEARO, Jakarta, 0612076 Multiclass, multiresidue analysis of pesticide in food is a major challenge to the analytical chemist. Why? Because of relatively great number and diversity of pesticide analytes and sample types. Pesticide Group? Fungicides, Herbicides, Insecticides Chemical Class? Dithiocarbamate, Imidazole, Thiocarbamate, Uracil, Carbamate, Organochlorine, Organophosporus, etc? Representatives of the chemical class? Ferbam, Ziram, Benomyl, Thiabendazole, Butylate, Cycloate, Bromacil, Lenacil, Aminocarb, Carbofuran, Dieldrin, Lindane, Chlorpyrifos, Diazinon, etc. Commodity? Orange, Apple, Grape, Lettuce, Chinese cabbage, wheat Because of relatively great number and diversity of pesticide analytes and sample types. Pesticide Group? Fungicides, Herbicides, Insecticides Chemical Class? Dithiocarbamate, Imidazole, Thiocarbamate, Uracil, Carbamate, Organochlorine, Organophosporus, etc? Representatives of the chemical class? Ferbam, Ziram, Benomyl, Thiabendazole, Butylate, Cycloate, Bromacil, Lenacil, Aminocarb, Carbofuran, Dieldrin, Lindane, Chlorpyrifos, Diazinon, etc. Commodity? Orange, Apple, Grape, Lettuce, Chinese cabbage, wheat

7. Dedi FardiazWHO SEARO, Jakarta, 0612077 Food Matrices Foods are often complex matrices with widely varying compositions. But, many foods consist of 4 major components: - Carbohydrates - Lipids - Proteins - Moisture Great variety of foods limits practicality to validate new analytical methods for all food matrices. For this reason, certain type of foods are proposed as a reference for other type of foods of similar nature. Example: Dutch monitoring program chooses orange, apple, grape, lettuce, Chinese cabbage, and wheat as representative commodities for validation studies.

8. Dedi FardiazWHO SEARO, Jakarta, 0612078 Some examples of how far the validation data may be extended to other commodities are: cereals, validation for whole grains cannot be taken to apply to bran or bread but validation for wheat grain may apply to barley grain or wheat four; animal products, validation for muscle should not be taken to apply to fat or offal but validation for chicken fat may apply to cattle fat; fruit and vegetables, validation for a whole fresh product cannot be taken to apply to the dried product but validation for cabbages may apply to Brussels sprouts.

9. Dedi FardiazWHO SEARO, Jakarta, 0612079 The Analytical Process Collection of a meaningful sample Transportation and storage of the sample Sample preparation: - Homogenization and subsampling -Extraction -Cleanup -Concentration Analysis (quantitation and confirmation) Data processing and quality review Reporting of the results Each step in the process must be carefully optimized

10. Dedi FardiazWHO SEARO, Jakarta, 06120710 The first consideration in choosing an analytical method is to define the needs for analysis. Once a list of the analytes and matrices has been prepared, then one should set the desired analytical figures of merit, which are: - limit of detection - recoveries - reproducibility ? 70 – 120% recoveries ? < 20 to 25% relative standard deviations ? < 10 to 100 ng/g

11. Dedi FardiazWHO SEARO, Jakarta, 06120711 Extraction of pesticides from the sample matrix is a fundamental aspect in the analytical process. Main factors in the extraction process: - sample matrix - extraction solvent, method, and time of extraction - sample:solvent ration - types and amount of salt added - comminution - water content - pH - temperature - pressure Each of these factors can have an effect on pesticide recoveries, stability and selectivity in the extraction, and these effects on the method being used should be known by the analyst.

12. Dedi FardiazWHO SEARO, Jakarta, 06120712 GUIDELINES ON GOOD LABORATORY PRACTICE IN RESIDUE ANALYSIS CAC/GL 40-1993, Rev.1-2003 The Guidelines are intended to assist in ensuring the reliability of analytical results in checking compliance with maximum residue limits of foods moving in international trade. Reliable analytical results are essential to protect the health of consumers and to facilitate international trade. The Guidelines are intended to assist in ensuring the reliability of analytical results in checking compliance with maximum residue limits of foods moving in international trade. Reliable analytical results are essential to protect the health of consumers and to facilitate international trade.

13. Dedi FardiazWHO SEARO, Jakarta, 06120713 ISO/IEC 17025 Standard OECD Good Laboratory Practice Principles Describes the requirements for: facilities management personnel quality assurance and quality control documentation of results and raw data and relevant subjects ISO/IEC 17025 Standard OECD Good Laboratory Practice Principles Describes the requirements for: facilities management personnel quality assurance and quality control documentation of results and raw data and relevant subjects These are considered as prerequisites for obtaining reliable and traceable results

14. Dedi FardiazWHO SEARO, Jakarta, 06120714 The Analyst (Section 2) Basic Resources (Section 3) The Analysis (Section 4) The Analyst (Section 2) Basic Resources (Section 3) The Analysis (Section 4)

15. Dedi FardiazWHO SEARO, Jakarta, 06120715 Residue analysis consists of a chain of procedures, most of which are known, or readily understood, by a trained chemist, but because the analyze concentrations are in the range µg/kg to mg/kg and because the analyses can be challenging, attention to detail is essential. The analyst in charge should have an appropriate professional qualification and be experienced and competent in residue analysis. The analyst must have an understanding of the principles of pesticide residue analysis and the requirements of Analytical Quality Assurance systems. Residue analysis consists of a chain of procedures, most of which are known, or readily understood, by a trained chemist, but because the analyze concentrations are in the range µg/kg to mg/kg and because the analyses can be challenging, attention to detail is essential. The analyst in charge should have an appropriate professional qualification and be experienced and competent in residue analysis. The analyst must have an understanding of the principles of pesticide residue analysis and the requirements of Analytical Quality Assurance systems. The Analyst

16. Dedi FardiazWHO SEARO, Jakarta, 06120716 The laboratory and its facilities must be designed to allow tasks to be allocated to well-defined areas where maximum safety and minimum chance of contamination of samples prevail. The area used for extraction and clean-up must meet solvent laboratory specifications and all fume extraction facilities must be of high quality. Use only small volumes of solvents the working area and store the bulk of the solvents separately, away from the main working area. Whenever possible, minimize the use of highly toxic solvents and reagents. Store all waste solvent safely and dispose safely and in an environmentally friendly manner. The laboratory and its facilities must be designed to allow tasks to be allocated to well-defined areas where maximum safety and minimum chance of contamination of samples prevail. The area used for extraction and clean-up must meet solvent laboratory specifications and all fume extraction facilities must be of high quality. Use only small volumes of solvents the working area and store the bulk of the solvents separately, away from the main working area. Whenever possible, minimize the use of highly toxic solvents and reagents. Store all waste solvent safely and dispose safely and in an environmentally friendly manner. The Laboratory

17. Dedi FardiazWHO SEARO, Jakarta, 06120717 All equipment such as lights, macerators and refrigerators should be “spark free” or “explosion proof”. Extraction, clean-up and concentration steps should be carried out in a well ventilated area, preferably in fume cupboards. There should be an ample supply of safety glasses, gloves and other protective clothing, emergency washing facilities and a spillage treatment kit. Adequate fire fighting equipment must be available. Staff must be aware that many pesticides have acutely or chronically toxic properties and therefore, great care is necessary in the handling of standard reference compounds. All equipment such as lights, macerators and refrigerators should be “spark free” or “explosion proof”. Extraction, clean-up and concentration steps should be carried out in a well ventilated area, preferably in fume cupboards. There should be an ample supply of safety glasses, gloves and other protective clothing, emergency washing facilities and a spillage treatment kit. Adequate fire fighting equipment must be available. Staff must be aware that many pesticides have acutely or chronically toxic properties and therefore, great care is necessary in the handling of standard reference compounds.

18. Dedi FardiazWHO SEARO, Jakarta, 06120718 Equipment and Supplies The laboratory will require adequate, reliable, supplies of electricity and water. Adequate supplies of reagents, solvents, gas, glassware, chromatographic materials, etc., of suitable quality are essential. Chromatographic equipment, balances, spectrophoto- meters etc. must be serviced and calibrated regularly and a recorded. Equipment used must be fit for purpose. All laboratories require pesticide reference standards of known and acceptably high purity. Care must be taken that standard solutions of pesticides are not decomposed by the effect of light or heat during storage or become concentrated by solvent evaporation. The laboratory will require adequate, reliable, supplies of electricity and water. Adequate supplies of reagents, solvents, gas, glassware, chromatographic materials, etc., of suitable quality are essential. Chromatographic equipment, balances, spectrophoto- meters etc. must be serviced and calibrated regularly and a recorded. Equipment used must be fit for purpose. All laboratories require pesticide reference standards of known and acceptably high purity. Care must be taken that standard solutions of pesticides are not decomposed by the effect of light or heat during storage or become concentrated by solvent evaporation.

19. Dedi FardiazWHO SEARO, Jakarta, 06120719 The Analysis Avoid Contamination One of the significant areas in which pesticide residue analysis differs significantly from macro-analysis is that of contamination and interference. They can give rise to errors such as false positive or false negative results. All glassware, reagents, organic solvents and water should be checked for possible interfering contaminants before use, by analysis of a reagent blank. Avoid Contamination One of the significant areas in which pesticide residue analysis differs significantly from macro-analysis is that of contamination and interference. They can give rise to errors such as false positive or false negative results. All glassware, reagents, organic solvents and water should be checked for possible interfering contaminants before use, by analysis of a reagent blank.

20. Dedi FardiazWHO SEARO, Jakarta, 06120720 Reception and Storage of Samples Identify the sample upon receipt and store it in a correct storage (if needed) If samples cannot be analyzed immediately but are to be analysed quickly, they should be stored at (1 - 5 °C), away from direct sunlight, and analyzed within a few days. However, samples received deep-frozen must be kept at -16 o C until analysis. In some instances, samples may require storage for a longer period before analysis. In this cases, storage temperature should be approximately - 20 °C, at which temperature enzymic degradation of pesticide residues is usually extremely slow Reception and Storage of Samples Identify the sample upon receipt and store it in a correct storage (if needed) If samples cannot be analyzed immediately but are to be analysed quickly, they should be stored at (1 - 5 °C), away from direct sunlight, and analyzed within a few days. However, samples received deep-frozen must be kept at -16 o C until analysis. In some instances, samples may require storage for a longer period before analysis. In this cases, storage temperature should be approximately - 20 °C, at which temperature enzymic degradation of pesticide residues is usually extremely slow

21. Dedi FardiazWHO SEARO, Jakarta, 06120721 Standard Operating Procedures (SOPs) Use Standard Operating Procedures (SOPs) for all operations. Any deviations from a SOP must be recorded and authorised by the analyst in charge. Validation of Methods Validation is the process of verifying that a method is fit for the intended purpose Standard Operating Procedures (SOPs) Use Standard Operating Procedures (SOPs) for all operations. Any deviations from a SOP must be recorded and authorised by the analyst in charge. Validation of Methods Validation is the process of verifying that a method is fit for the intended purpose

22. Dedi FardiazWHO SEARO, Jakarta, 06120722 BIOLOGICAL HAZARDS CHEMICAL HAZARDS PHYSICAL HAZARDSFREE HAZARDS SAFE THE FOOD and FREE THE PRODUCT from HAZARDOUS SUBSTANCE SAFE THE FOOD and FREE THE PRODUCT from HAZARDOUS SUBSTANCE Thank You Very Much