PESTICIDE RESIDUE AN4LTiSIS 37 Pesticide Residue Analysis The following paper originated from a discussion at a meeting of the ad hoc Working Group on Methods of Analysis of the Codex Committee on Pesticide Residues in The Hague, Holland, 1977. I t was discussed in draft form a t the 1978 meeting of the Working Group and by the UK Committee on Analytical Methods for Pesticide Residues (CAM), and also at the meeting of the IUPAC Commission on Pesticide Residues Analysis in Deidesheim, West Germany, 1978.The author acknowledges the help given by many colleagues, including members of the above bodies, in producing these guidelines.38 PESTICIDE RESIDUE ANALYSIS Proc. Analyt. Div. Chem. SOC. Good Analytical Practice in Pesticide Residue Analysis G. M. Telling Unilever Research Laboratory, Colworth House, Shavnbvook, Bedfordshire, MK44 1LQ The Codex document Alinorm 76/24 Appendix IV (Report of the ad hoc Working Group on Methods of Analysis) contained the following statement.“It was considered that the ultimate goal in fair practice in international trade depended, among other things, on the reliability of analytical results. This in turn, particularly in pesti- cide residue analysis, depended not only on the availability of reliable analytical methods, but also on the experience of the analyst and on the maintenance of ‘good practice in the analysis of pesticides.’ ” This paper is an attempt to define such good analytical practice and may be considered in three inter-related parts: The Analyst Basic Resources The Analysis.The Analyst Residue analysis consists of a chain of procedures, most of which are known, or readily understood, by a trained chemist, but because the margin of error is smaller than in most other types of analysis and any mistake can invalidate the whole analysis, attention to detail in thes2 procedures is essential. There should be adequate overlap and continuity of staff and all need to be experienced in residue analysis over a period of years.Staff should be trained in the correct use of apparatus and basic laboratory skills and the basic principles of residue analysis. They must understand the purpose of each stage in the method being used and the importance of following the method exactly as described and of noting any enforced deviations.A clear understanding of the terminology involved is also essential. Ideally, when a laboratory for residue analysis is set up, the staff should spend some of their training period in a well established laboratory where experienced advice and training is available. If the laboratory is to be involved in the analysis for a wide range of pesticide residues it may be necessary for the staff to gain experience in more than one established laboratory.Basic Resources The Laboratory In ideal circumstances the laboratory and its fittings should be designed to allow tasks to be allocated to well defined areas with maximum safety and minimum chance of contamination of samples. Fittings should be of materials resistant to attack by chemicals likely to be used in the area.Thus, in such ideal conditions separate rooms would be available for sample re- ceipt and storage, for sample preparation, for extraction and clean-up and for instrumentation used in the determinative step. The area used for extraction and clean-up would meet solvent laboratory specifications and all fume extraction facilities would be of high quality. The minimum requirements for pesticide residue analysis are that the facilities are adequate to avoid contamination.Laboratory safety must also be considered in terms of necessary and preferable conditions as it must be recognised that the stringent working conditions enforced in residue laboratories in some parts of the world would be totally unrealistic in others. No smoking, eating, drink- ing or application of cosmetics should be permitted in the working area.Only small volumes of solvents should be held in the working area and the bulk of the solvents stored separately, away from the main working area. The use of toxic solvents and reagents must be avoided whenever possible. The main working area should be treated as a solvent laboratory and all equipment such as lights, macerators and refrigerators should be spark-free.Extractions, clean-up and con- centration steps should be carried out in a well ventilated area, preferably in fume cupboards or under fume hoods. All waste solvents should be stored safely and disposed of frequently.January, 1979 PESTICIDE RESIDUE ANALYSIS 39 Safety screens should be used when glassware is used under vacuum or pressure.There should be an ample supply of safety glasses, gloves and other protective clothing, emergency washing facilities and spillage treatment kit. All staff should be trained in the use of these facilities and in an appreciation of the hazards involved. Staff must be aware that many pesticides have toxic and/or carcinogenic properties and although little risk is attached to the handling of most samples, great care is necessary in the handling of standard reference com- pounds.Adequate fire fighting equipment must be provided. The staff should be given periodic medical checks. Equipment and Supplies Supplies The laboratory will require adequate supplies of electricity and water and various gases, either piped or from gas cylinders of proven quality. Adequate supplies of reagents, solvents, glassware, stationary phases, etc., are essential.Servicing facilities for gas chromatographs, balances, spectrophotometers, etc., will be required and will probably involve keeping some essential spare parts plus access to a good technical service. Adequate equipment Although, in an ideal situation, equipment should be regularly updated in order to keep up with developments, e.g., gas chromatography with microprocessor controls, the equipment only needs to be sophisticated enough to do the job required. Thus, the demands for monitor- ing commodities a t tolerance levels laid down in the Codex are much less stringent than those required in a research environment.All laboratories require an adequate range of standard pesticides of known and reasonably high purity.The range should cover all parent species for which the laboratory is moni- toring samples as well as their more common metabolites. The Analysis Avoidance of Contamination One of the major areas in which pesticide residue analysis differs significantly from macro- analysis is that of the problem of contamination. Trace amounts of contamination in the final samples used for the determination stage of the method can give rise to errors such as false positive results and to a loss of sensitivity that may prevent the residue analyst from achieving the necessary limits of determination. Contamination may arise from either the environment or the procedure.Contamination from the working environment Bench polish, barrier creams, soaps containing germicides, fly sprays, perfumes and cos- metics are all commodities that can give rise to laboratory contamination and are especially significant when an electron-capture detector is being used.There is no real solution to the problem other than to ban their use. Greases, plasticisers, rubber bungs and tubing, oil from air lines, extraction thimbles, filter-papers and cotton-wool can also all give rise to contamination of the final test solution.Pesticide reference standards should always be stored in a room separate from the main residue laboratory. Field samples, sample preparation and formulation analysis should also be kept separate from the main residue laboratory. Contamination from the procedure being used previous samples.then rinsed with the solvent to be used. pesticide residue work. contain components that interfere in the analysis. adsorbents by heating and it is generally necessary to use redistilled solvents. Contamination of glassware, syringes and gas-chromatographic columns can arise from All glassware should be cleaned with detergent, rinsed thoroughly and There must be a separate stock of glasswarefor Chemical reagents, adsorbents and general laboratory solvents may It may be necessary to purify reagents and De-ionised40 PESTICIDE RESIDUE ANALYSIS Proc.Analyt. Div. Chew. SOC. water is often suspect and redistilled water is preferable. In many instances tap water or well water may be satisfactory. Other materials containing plasticisers are suspect but PTFE and silicone rubbers are usually acceptable and others may be acceptable in certain circumstances.Sample storage containers can cause contamination and glass bottles with ground glass stoppers should always be used. Instru- mentation should always be housed in a separate room. The nature and importance of con- tamination can vary according to the type of determination technique used and the level of pesticide residue to be determined. These contamination problems, which are important with methods based on gas chromatography or HYLC, may well be less significant if a spectro- photometric finish is used, and vice versa.For relatively high levels of residues the background interference from solvents and other materials may be insignificant in comparison with the amount of residue present, while many problems can be solved by the use of specific detectors.Furthermore, if the contaminant does not interfere with the residue being sought, its presence may be acceptable. No apparatus containing PVC should be allowed in the residue laboratory. Avoidance of Losses Losses during storage In an ideal situation samples should be stored at chill temperature, away from direct sun- light, and analysed within a few days.However, in many instances samples can require storage for an extended period (6-9 months) before analysis and the following precautions should be observed. Storage temperature should be approximately -20 O C , when degradation of residues of pesticides by enzyme action is extremely slow.If any doubts exist, the samples should be compared with fortified samples stored under the same conditions. All samples should be re-homogenised after freezing as there is a tendency for water to distil out and to collect as ice crystals, which, if discarded, will affect the analytical result. Neither the containers used for storage nor their caps or stoppers should allow migration of the chemical being sought into the container.The containers must not leak. All samples should be labelled clearly with permanent labels and recorded in a sample book. Losses dutring the analysis The extracts and final test solutions should not be exposed to direct sunlight. Validation of Methods In a routine laboratory monitoring for compliance with Codex or national tolerances, standardised methods will be used in most instances and effort expended on validation of methods will be at a minimum.In all laboratories, regular checks will be made on the effects of variation in sources of supply of chemicals, solvents, etc. The performance of the method will have to be checked by, for example, the recovery of standards, added at appropriate levels, taken through the method both alone and in the pres- ence of each new substrate.The effects of light, storage at intermediate stages of the procedure, temperature, etc., on the stability of reagents and samples must be studied. The evaluation of detection/determination systems (e.g., in gas or liquid chromatography) for effects of flow-rate, temperature, etc., is important. In laboratories where method development and/or modification is undertaken other aspects that may be studied are the effect of variation in sample size, partition ratios, etc., theefficiency, resolution and column stability of gas- and liquid-chromatographic systems and variations in activity of various column clean-up systems.The amount of effort allocated to the validation of methods will vary considerably.Maintenance of Over-all Analytical Performance of the methods in use, both at the tolerance level and at the lower limit of determination. In all laboratories engaged in pesticide residue analysis there is a need for regular assessmentJ a m a r y , 1979 PESTICIDE RESIDUE ANALYSIS 41 Rwovery studies Recovery of pesticides from “spiked” samples is commonly used as a measure of efficiency of extraction, but it must be recognised that such studies are of limited value.More emphasis should be placed on checking recoveries where residues are in a “real” state, e.g., in aged samples. I t must also be recognised that a method that gives adequate recoveries from samples spiked with parent compounds may be inadequate for the measurement of significant metabolites produced during ageing of the substrate.Recoveries should be within the range ‘iO-llO(?b with a mean of greater than SOo,b after removal of outliers. Bluii k responses and interferences check that contamination is not occurring. Regular analyses of substrates known to be free of pesticide residues is necessary in order to Stubilitv of standards Regular injection of standards during the analysis of a series of samples allows the perform- ance of the determination step to be checked.In addition, care should be taken that standard solutions of pesticides are not decomposed by the effect of light or heat during storage or become more concentrated owing to solvent evaporation. Equal care must be taken to ensure t lie stability of reference standard compounds.-4 Irnljisis of check samples check samples at regular intervals. samples without any indication being given as to their special nature. ;ln excellent means of monitoring the performance of a method (or an analyst) is to introduce These check samples should be introduced as routine Participation in collaborative studieslring tests I’arious national and international organisations now organise collaborative studies on particular methods and/or ring tests on particular substrates.These present an ideal way for laboratories to assess their own performance. If possible, collaborative samples should be introduced as routine samples so that the analyst concerned does not attempt to “make a special effort,” which would invalidate the samples as a test of laboratory performance.Confirmatory Tests beforehand, confirmatory tests will not normally be necessary. the reliability of results need confirmatory tests be used. under a number of headings. Use of solvent partitioning effects such as P values. Use of multiple gas-chromatographic columns. Use of different chromatographic techniques. For routine control, where the range of resulting values is, at least to a certain extent, known Only if there are doubts as to Confirmatory tests can be considered Although this technique is widely used, its value is limited because, in all instances, the basic chromatographic technique is similar.In many instances confirmation of gas- chromatographic findings is best achieved by using thin-layer chromatography or high- Performance liquid chromatography.Both have considerable advantages over gas chromato- graphy in some circumstances, especially when dealing with substances that are not thermally stable. Whenever possible, confirmatory techniques should be carried out rather than placing complete reliance on gas-chromatographic columns as a method of identification. Use of different detector systems.I‘se of chemical derivatisation techniques. These are widely used techniques and a number of text books are available on the types of derivatisation that can be achieved. A closely linked technique is the use of, for example, ultraviolet light to change the chemical structure of the compound under examination. Gas chromatography - mass spectrometry is a technique widely used in laboratories with a high level of sophisticated instrumentation, although it is not available in the majority of pesticide residue laboratories.Pre-gas - liquid chromatographic separation techniques often give an indication of the identity of residues, as they are based on the properties of residues present.42 ANALYTICAL CHEMISTRY TRUST FUND Proc.Analyt. Div. Chem. sot. Reporting Results This aspect of pesticide residue analysis depends very much on the requirements of the organisation demanding the analytical information and it is difficult to lay down strict rules of reporting, or even on the accuracy required. I t is recommended that both analyst and user of the information fully appreciate the capability of the methods used and the interpretation to be placed upon data produced before the work is started. In all instances reports of results should indicate the method used in order to obtain those results. I acknowledge the help that I have received from numerous colleagues who were prepared to discuss what they thought such a document should contain. Bibliography Burke, J., and McMahon, B., “Analysis of Food for Residues of Pesticides,” FDA By-Lines, No. 4, January “Guidelines on Analytical Methodology for Pesticide Residue Monitoring,” Federal Working Group on Pest Sherma, J , , “Manual of Quality Control for Pesticides and Related Compounds in Human and Environ- “Pesticide Analytical Manual,” Volume 1 , U.S. Department of Health, Education and Welfare, Food and 1977. Management, Washington, D.C. 20460, June, 1975. mental Samples,” USA Environmental Protection Agency, EPA 600/1-76-017, February, 1976. Drug Administration.