Pesticide Residue Method Development and ... - ACS Publications

Chapter 12

Pesticide Residue Method Development and Validation at the Food and Drug Administration

Downloaded by MONASH UNIV on November 18, 2015 | http://pubs.acs.org Publication Date: December 31, 1991 | doi: 10.1021/bk-1991-0446.ch012

Marion Clower, Jr. Pesticide and Industrial Chemicals Branch, Division of Contaminants Chemistry, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 200 C Street SW, Washington, DC 20204 The Food and Drug Administration's (FDA) responsibility for monitoring the nation's food supply for pesticide residues is the driving force behind FDA's pesticide residue method development. Efficiency dictates that multiresidue methods, which can analyze simultaneously for many residues in a variety of agricultural products, be used in and developed for such monitoring. Methods that analyze for a single or small number of residues are employed on a limited basis when necessary. Overall method development activities are guided by a Five-Year Plan, which is updated annually to reflect accomplishments and new objectives. Method development activities focus on extension and expansion of existing methods to additional pesticides and crops. New analytical techniques are investigated for applicability to existing and new methods. Studies of the analytical behavior of pesticide chemicals in existing methods play an important role in method research. A computerized database tracks capabilities of FDA multiresidue methods and assists in guiding new method development. Before use in the monitoring program, all methods are validated in intralaboratory and interlaboratory studies to assure that they perform properly and provide reliable analytical information. Collaborative studies are conducted under the auspices of the Association of Official Analytical Chemists. The Environmental Protection Agency (EPA) registers (approves) the use of pesticides and, in cases where residues might occur on foods, establishes tolerances (maximum residue limits). FDA is responsible for enforcing these tolerances for pesticide residues in foods, except meat and poultry, which are the responsibility of the Department of Agriculture. The nationwide monitoring program resulting from this responsibility requires development of pesticide residue methods in FDA. This chapter not subject to U.S. copyright Published 1991 American Chemical Society

In Pesticide Residues and Food Safety; Tweedy, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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FDA Monitoring Program FDA monitors the food supply, using two complementary approaches. Regulatory monitoring is specifically designed to enforce tolerances and other regulatory limits for foods in interstate commerce and those offered for import into the United States. Emphasis is placed on analysis of a wide variety of raw agricultural commodities for large numbers of pesticide residues. Regulatory monitoring is also the principal source of information on the incidence and level of pesticide residues in the general food supply. Such foods are analyzed in almost a dozen FDA laboratories across the country. FDA also conducts a Total Diet Study to monitor dietary intakes of pesticide residues and to identify trends in residue levels. Samples collected from retail markets in representative areas of the country are analyzed in a single laboratory after being prepared as if for consumption. Accurate determination of residue levels is clearly necessary for tolerance enforcement, but is also essential for calculation of dietary intakes even when residue levels are below applicable tolerances. Therefore, both monitoring approaches require quantitative analytical methods. Methods must also be applied uniformly in all laboratories. Types of Analytical Methods The large and ever-growing number of pesticides available for use on foods and the unknown treatment history of many samples collected in FDA's monitoring program have caused FDA research to concentrate on developing methods that can identify and measure more than one residue in a single analysis. These general purpose multiresidue methods, or MRMs, are widely used because they provide coverage for a large number of residues of different chemicals and are usually applicable to many different commodities, thus allowing the most efficient use of resources. Recently registered pesticides are often not determined by these general purpose MRMs because their physical properties are radically different from those for which the MRMs were developed. A selective multiresidue method, specifically designed to recover a small group of structurally similar pesticides, must then be employed. Although selective MRMs recover multiple residues, the number is not large—usually less than 20. These methods are also frequently more complicated or they may require recently developed, sophisticated instrumentation. Single residue methods, or SRMs, are usually capable of determining the residue of only one pesticide from a limited number of commodities. SRMs are usually submitted during the registration process to meet EPA's requirement for an analytical method capable of determining compliance with the requested tolerance. SRMs are used in FDA monitoring when data on a specific residue are needed and no general or selective MRM is available.



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Pesticide Residue Method Development and Validation at FDA 107

Most methods have limitations in one or more of the following attributes: coverage (residues and foods), speed, complexity, and expense. SRMs are the most limited since they are capable of determining only one residue. SRMs are often lengthy, require specialized equipment, and in many cases are difficult to perform. SRMs are typically developed for a specific purpose and are used for a limited time. Their limitations often appear less important or restrictive when the method is familiar and meets an immediate need. Compared to SRMs, selective and general purpose MRMs have broader coverage, but also suffer to varying extents from the same limitations. The cleanup step, in particular, generally requires a large fraction of analysis time for any method. The typical "macro" design (100 g analytical portion) of most existing methods makes them expensive and time-consuming. Aspects of Analytical Method Development The potential use of a number of structurally different pesticides on the same food presents a problem for analytical methodology that is based on a common chemical or physical property of all target chemicals. Many newer pesticides are very polar relative to older ones, making them less amenable to determination by existing methods. Most modern pesticides give rise to metabolites that differ substantially in structure from the parent pesticide. This difference often requires different methodology for parent and metabolite(s). When the tolerance applies to the "total residue", several methods must frequently be used. Overall, the method must be applicable to the appropriate foods, be capable of quantitating residues of interest below the tolerance level (or lower in the Total Diet Study), and provide an extract suitable for confirmation of residue identity. These challenges to development of analytical methods are met by conducting research that will be most beneficial and productive for the monitoring program. Analytical method studies usually focus on the following activities: extension of an existing method to include additional analytes; expansion of a method to cover new foods; integration of new technology into an existing method; validation of a method, technique or modification; and development of a new method or technique. The first four of these activities are part of the development of a comprehensive MRM. The large and increasing number of pesticides and metabolites with tolerances on a wide array of foods results in the possibility of many different analytical situations. Incorporation of new technology into existing methods and those under development is the principal means of providing comprehensive analytical methodology. Validation of all improvements is essential to ensure credibility of resulting data. These five activities are organized to produce a suitable analytical approach as efficiently as possible.



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Steps in Method Development Logically, before any method research begins, the structure and chemical nature of the target chemicals must be reviewed to evaluate the possibility of recovery by an existing method. If this review indicates, recovery studies are conducted with one or more of FDA's five principal MRMs. MRMs are still the most effective way to examine foods of unknown treatment history. For pesticides registered in the United States, or when uses of a pesticide registered in foreign countries are known, recovery studies will involve the appropriate agricultural commodities. Successful recovery (usually 80-120%) generally eliminates the need for method research since the pesticide will be covered in FDA's monitoring whenever that MRM is employed. In situations where structure review is not definitive, a procedure has been developed for testing the analytical behavior of a pesticide. This protocol is a decision tree which guides method testing through allfiveFDA MRMs in a logical manner. The decision tree assures that only appropriate method testing is conducted. For example, only pesticides containing an Nmethyl carbamate group should be tested through FDA's carbamate method since it can determine chemicals with that moiety only. In addition to FDA laboratories, the decision tree has been provided to EPA's Dietary Exposure Branch for use by petition reviewers who communicate with registrants about EPA requirements. EPA has distributed copies to registrants who intend to perform method behavior tests. Protocols for testing analytical behavior of pesticides have been developed for each of FDA's five MRMs. All protocols, which include standardized reporting forms to ensure consistent, appropriate data suitable for entry into FDA's analytical behavior data tracking system, are compiled as Appendix II in Volume I of FDA's Pesticide Analytical Manual (PAM) (7). In this way, the most useful analytical data will be obtained in an efficient manner. For pesticides not recovered by existing MRMs, selective MRMs are investigated next. A suitable selective MRM can often be efficiently developed by minor modification (e.g., a new or different detector) of an existing general or selective MRM. New MRMs often have their basis in existing SRMs or parts of methods published in the literature. In the absence of other information, the first SRMs evaluated are those contained in Volume II of the PAM. These methods have been submitted to EPA as part of the pesticide registration process. When a method is needed immediately, or method research time is otherwise unavailable, the analytical capability of an existing SRM is evaluated. A protocol has also been developed for selection and evaluation of residue methods in general and is applicable to SRMs. This protocol was published as FDA Laboratory Information Bulletin 3216 (2) and contains information similar to that in the MRM protocols. The directions, however,



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have been generalized to allow evaluation a method for use in a survey of any specific residue/food combination. The protocol describes a logical sequence of steps to efficiently determine the most appropriate method for evaluation on the basis of the method's intended use, which must be clearly defined before laboratory activities begin. A computerized database, Pestrak, has been developed to track results from tests of the analytical behavior of chemicals in FDA's MRMs. Data for SRMs are also included when this information is known for chemicals that are not recovered by an MRM. Pestrak contains information on about 1000 pesticide chemicals—pesticides, metabolites, and degradation products—including those registered in the United States and many registered in foreign countries. Many of these chemicals currently have no significance for study, because they are no longer used, but are being tracked to maintain an awareness of all potential pesticide chemicals for which methods may be needed. Pestrak contains additional information that gives a thumbnail sketch of each chemical. For example, codes are assigned to indicate the status of chemicals undergoing Special Review by EPA. Pestrak also contains information that indicates the most appropriate method test that should be applied to each chemical. Since Pestrak is computerized, it can be searched according to user-definable criteria for optimum selection of a chemical or group of chemicals for simultaneous method testing or development. Many Pestrak chemicals for which analytical behavior data are unavailable are being studied in FDA laboratories as part of continuing MRM expansion studies. A number of these have been targeted for study, but laboratory work cannot begin because analytical reference standards are unavailable. In 1986, EPA began requiring that registrants include data on the analytical behavior of residues through FDA's MRMs in registration petitions. Information for over 70 such chemicals has been incorporated into Pestrak. Current Methods Development Research Within FDA The Pesticides and Industrial Chemicals Branch, in the Center for Food Safety and Applied Nutrition, is the FDA headquarters unit responsible for pesticide residue methods research. The overall effort, however, includes the Pesticides and Industrial Chemicals Research Center (PICRC), the Total Diet Research Center (TDRC), and Field laboratories, which are principally involved in pesticide residue monitoring. The following discussion highlights current methods research and describes how this research is coordinated into a cohesive research program. Numerous projects designed to improve FDA's methodology through incorporation of new or improved analytical techniques are also conducted on a continuing basis in all FDA laboratories.



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Anilines—A number of important pesticides or metabolites have a substituted aniline or nitroaromatic structure and cannot be determined by current methods. The electrochemical detector used in high pressure liquid chromatography (HPLC) provides a convenient way to measure such compounds by virtue of its ability to oxidize or reduce a chemical passing through the detector. Conditions for determination of a group of anilinecontaining pesticide chemicals have been developed using this detector. Because of its widespread use, alachlor, a typical compound from this group, is the focus of current research. Once this method is developed, it can be readily adapted to determination of about 15 additional anilinecontaining residues. Immunoassay kits—Commercial immunoassay detection kits are generally suitable for use in SRMs since the antibody involved reacts with a single pesticide. One kit presently available, however, detects several common triazines—atrazine, ametryn, prometryn, and simazine. This kit is being evaluated for detection of triazines in a food matrix extract that is typical of those obtained in FDA's MRMs. Immunoassay methods—FDA is also developing, by contract, six pesticide residue methods based on enzyme immunoassay technology. Methods will be evaluated by the same criteria that are applied to traditional residue methods. Each phase of the contract was designed to obtain specific information on the applicability of this technique to pesticides that present unusual difficulties in traditional methods. In Phase I, a method will be developed for determination of paraquat in potatoes. Since paraquat is a small water-soluble molecule, Phase I will test the applicability of immunoassay techniques to small molecules such as pesticides. Phase II will examine the multiresidue capability of immunoassays by developing a single assay for a parent pesticide (fenamiphos) and two of its metabolites (fenamiphos sulfoxide and fenamiphos sulfone) in oranges. Development of separate immunoassays for carbendazim, benomyl, and thiophanate-methyl in apples (Phase III) will provide a unique challenge, not only because these pesticides are structurally similar, but also because analysis by traditional residue methods is hampered by chemical instability of these compounds in organic solvents. A method for glyphosate in soybeans will be developed in Phase IV. Glyphosate contains an amino acid moiety, which may hamper detection by an immunoassay. Use of soybeans as the food matrix will suggest potential problems with immunoassay of fatty foods. Quaternary amines—Development of a selective MRM for paraquat, diquat, difenzoquat, mepiquat, and chlormequat began last year. These pesticides present an analytical challenge because of their very polar nature; techniques used in traditional MRMs must be radically modified to be applicable. Total Diet Study methods—TDRC concentrates on methods that overcome the special difficulties in analysis of the cooked foods examined in the Total Diet Study. These methods must also be more sensitive (generally 5to 10-fold) than regulatory methods, to quantitate low levels of residues



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found and thereby provide finite estimates of dietary intake. Techniques and methods developed by TDRC are also applicable to the pesticide monitoring program. Robotics and automation—TDRC is extensively involved in evaluation and adaptation of new or improved analytical techniques to both the general and selective MRMs. For example, TDRC is investigating use of automation and robotics in Total Diet Study methodology. Robotics is particularly applicable, since this is the only portion of FDA's pesticide program with the defined, repetitive sample load required for efficient use of robotics. Photodiode detector—In addition to method development research, analytical techniques are studied for applicability to existing, modified, or newly developed methods. A Field laboratory is evaluating the photodiode array detector with a selective MRM because of its capability to monitor HPLC column effluent at a number of different UV wavelengths simultaneously. Although UV detection is usually not sufficiently specific for positive identification of pesticide residues, it may prove useful as a screening tool. Validation of Analytical Methods

Validation of analytical methods is an integral part of the method development process. At a minimum, validation assures that the method directions are properly written and thus can be carried out by an experienced analyst. More importantly, validation assures that the method performs properly and provides accurate analytical information; the method, therefore, will be able to withstand potential legal challenges for accuracy and reliability. Validation also permits equivalent application of the method in multiple laboratories and promotes uniform enforcement monitoring. Method validation usually proceeds from intra- and interlaboratory studies within FDA to an AOAC collaborative study if appropriate. Once the method developer is satisfied with method performance, an intralaboratory study is conducted to provide assurance that the method and its written directions are generally usable. At this point, the method and its supporting data are often submitted for publication in the scientific literature, or in an FDA Laboratory Information Bulletin. These documents contain analytical methods and techniques and are published continuously by FDA as a means of providing rapid dissemination to all FDA laboratories of analytical techniques and methods that appear to work, but have not been thoroughly validated. Before a method is used in FDA monitoring, validation of its accuracy and precision in an interlaboratory trial is required. Usually two laboratories test the method by analysis of foods fortified with residues at levels unknown to the analysts. Crops withfield-incurredresidues are preferred, but are typically unavailable. Recovery experiments are conducted at least in duplicate, using foods fortified with the residue(s) at two or more levels.



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Fortification levels are selected to encompass a range of one or two orders of magnitude. The lower fortification level is often near the method's quantitation limit, but is always below the tolerance for the residue/crop combination. Analysis of a reagent blank (water replaces the crop matrix) and analysis of a crop blank (no residue) are also completed to assure the absence of interfering chemicals. Analytical results of such studies are expected to show recoveries between 80 and 120% of the fortified residue level. More rigorous method validation is undertaken for methods that prove to have widespread and continuous use—a collaborative study conducted under the auspices of the AOAC. Collaborative studies are usually undertaken for methods that determine residues of high regulatory interest or those for which FDA expects widespread use, but only after experience in many laboratories indicates that they are worth the considerable effort and cost involved. If the results of the collaborative study meet AOAC statistical requirements for accuracy and precision, the method is adopted as "official" and published in AOAC's Official Methods of Analysis (5). This publication and Volume I of FDA's PAM contain, by reference in the U.S. Code of Federal Regulations (4), methods which are official for regulatory use. The PAM has had numerous revisions since it was first issued in 1963. A chapter on HPLC will be included in the next revision, and a third edition of Volume I of the PAM will be prepared under contract in the near future. In addition to FDA's five MRMs, Volume I contains ancillary information on such topics as preparation of samples and standard reference solutions, and operation of the various instruments used in pesticide residue analysis. The five MRMs include three general purpose MRMs: the Luke method, section 232.4; the Mills fatty food method, section 211.1; and the Mills, Onley, and Gaither nonfatty food method, section 212.1. The other two methods are considered selective MRMs, i.e., the Storherr method for organophosphorus compounds, section 232.3; and the Krause method for N-methylcarbamates, section 242.2. The only other selective MRM currently included in Volume I is the Hopper method for chlorophenoxyacetic acids, section 221.1. Several general and selective MRMs that are used in FDA monitoring have not yet been added to Volume I since they have not undergone sufficient testing. The methods in Volume II of the PAM are principally SRMs, although some methods can recover several metabolites in addition to the parent pesticide. These methods, developed to measure residues identified in the tolerance expression, are generally submitted by pesticide registrants as part of the registration process. SRMs developed by FDA can be included in Volume II; however, FDA usually tests the applicability of existing Volume II methods rather than developing new ones. Results of these evaluations are included in the manual as "User Comments". Submission of such comments from laboratories that investigate or use such methods provides a better understanding of the capabilities of more of these methods.


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Summary


Analytical method development and validation are essential parts of FDA's program to monitor the nation's food supply and enforce regulatory limits for pesticide residues. Method development research, guided by a Five-Year Plan, is a cooperative effort conducted in all FDA laboratories involved in the monitoring program. Emphasis is placed on expansion and extension of existing multiresidue methods. New technology is incorporated into existing and newly developed methods to increase method capability and efficiency. Method validation, the final step in method development, assures that the analytical data produced are reliable and accurate. Literature Cited

1. Pesticide Analytical Manual, Vols. I and II, Food and Drug Administration: Washington, DC, 1986. 2. Parfitt, C. H. Laboratory Information Bulletin 3216, Food and Drug Administration: Rockville, MD, 1988. 3. Official Methods of Analysis; Williams, S., Ed., Association of Official Analytical Chemists: Arlington, VA, 1984. 4. Code of Federal Regulations; U.S. Government Printing Office: Washington, DC, Title 21, Part 2.19; Title 40, Part 180.6(d), 1989. RECEIVED

August 19, 1990


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