Rapid Test Methods for Regulatory Programs - ACS Symposium

Chapter 4

Rapid Test Methods for Regulatory Programs Richard L. Ellis

Downloaded by AUBURN UNIV on November 18, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch004

Food Safety and Inspection Service, Science and Technology Program, U.S. Department of Agriculture, 200 12th Street, Southwest, Cotton Annex, Washington, D C 20250

A major component of food safety programs is to assure compliance with regulatory limits for pesticides, environmental contaminants and veterinary drugs. While the Food and Drug Administration and the Environmental Protection Agency have primary responsibility for establishing regulatory limits for these substances, the U.S. Department of Agriculture (USDA) has the daily responsibility for determining compliance with these residue limits in meat and poultry products. This food safety responsibility is accomplished through USDA's National Residue Program, complimented on occasions with special residue studies. Meeting the objectives of a statistically designed residue control program that traditionally examines 10 or more classes of xenobiotics and more than 75 individual compounds requires a wide variety of analytical and microbiological methods and screening tests in inspection facilities and laboratories using animal tissue, biological fluids or other matrices as a test media. Environmental, economic, regulatory and evolving public health considerations will require new strategies with more focus on screening methods to complement traditional quantitative and confirmatory laboratory methods.

Immunochemistry based assays are emerging as promising screening test methods. Test systems for a wide variety of organic residues in soil, water, food, plant and animal tissues are being developed by scientists in the public and private sectors in the United States. Examples of new immunochemistry based tests are presented in other chapters of this book. In addition, organizations such as AOAC International have developed and implemented test kit evaluation programs (e.g., A O A C Research Institute) to assure test performance with the sponsor's labeling claims. These tests are being developed in rapid, very sensitive, easy and usually highly specific formats.Immuno-based assays presented at this ACS meeting show promise as rapid non-laboratory qualitative assays, while some are now being designed for fast, quantitative, laboratory tests. Their antibody design specificity, which is commonly very high, generally permits use of relatively simple This chapter not subject to U.S. copyright Published 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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sample preparation procedures for test materials and makes irnmunochernisry methods attractive for use in laboratory as well as non-laboratory environments. Generally, the cost of these assays is less than that of traditional analytical laboratory methods even though most of these test systems are dependent on some sample preparation. Nevertheless, per sample cost for such assays is usually less than 25 percent (including administrative costs) of costs for instrumental methods for similar analyses. The major constraint for many of these assay systems is their relatively high cost of development. A n Australian study (7) estimates that development of test kits for pesticide residue screening becomes practical when markets for 100,000 test kits per year or more are anticipated.


Method Performance Concepts An analysis scheme that can simultaneously quantify the presence of all compounds or classes of compounds of interest in foods, animal tissue or fluid with acceptable accuracy and correctly identify the analyte or analytes would be a desirable, unified approach for regulatory control agencies. However, at the present time there are very few analytical procedures available to regulatory agencies that can simultaneously quantitate and confirm the identity of such residues. Until universal methods are available, regulatory programs will have to employ methods with individual attributes of presumptive presence, quantification and positive identification. To satisfy this goal, methods with different attributes must integrate well with each other for a highly effective residue control program regardless of individual regulatory mandates. Terms such as confirmatory, reference, quantitative, or rapid methods are well known but may mean different things to different people. An alternative to categorizing methods with the potential paradigm (e.g., every one knows what confirmation means) associated with these descriptive terms, is to define the methods according to the attributes or qualities of method performance. Attributes and qualities for three types of analytical methods are relevant to support regulatory programs. While the focus is on scrœning methods, a brief description of the method types is intended to help clarify their interrelationship. A detailed description has been published recently in a Codex document developed by the Codex Committee on Residues of Veterinary Drugs in Food (2). Recently, the Codex Committee on Methods of Analysis and Sampling began considering this approach for other commodities. Type I methods have the ability to quantify the amount of a specific analyte or class of analytes and provide positive identification in a single analytical process. These are assays with the highest level of credibility for providing quantitation and unequivocal identification at the level of interest. They may be single procedures that determine the concentration and identity of the analyte, or combinations of procedures for determining analyte concentration and confirming its structure. Few such methods currently exist for use in regulatory programs. These methods often include a mass spectrometry component. Type II methods are not unequivocal for identification of an analyte at the concentrations of interest but are useful for determining the concentration of an analyte and providing some structural information. For example, these methods may employ structure, functional groups or biochemical properties (e.g., mechanism of action) as the

Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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basis for the analytical scheme. These methods are generally reliable enough to be used as regulatory reference methods and provide a very acceptable approach for residue control programs. These methods may also be used to corroborate the presence of a compound or class of compounds. Thus, two Type Π methods may provide information suitable for Type I attributes providing they employ different chemical technologies. There are two fundamental applications for analytical methods in regulatory analysis schemes. For situations where there is a regulatory tolerance or action limit, quantification around the regulatory limit is the primary consideration. Where no regulatory limit has been established for an analyte (the compound is not approved for use in food producing animals or birds) the primary issue is identification rather than quantification of the analyte. The more common scenario is quantification of an analyte. In the latter case, confirmation of sturctural identity is a critical concern. The majority of analytical methods presently available and used by regulatory control agencies are Type Π methods used for quantification. Type ΙΠ methods are those that generate less specific though useful information. These testing procedures, for example, detect the presence or absence of a compound or class of compounds at some designated level of interest and often are based on noninstrumental techniques for analytical determination. Screening methods are typically Type ΙΠ methods. An important consideration for Type ΙΠ methods is their use to rule out the presence of the analyte at or above a concentration of interest. Assuming that a Type HE method has an acceptable limit of false negative performance, no further analysis may be required. Samples that yield a positive response using a Type ΙΠ method require additional analysis before a regulatory disposition can be made. Thus, Type ΠΙ methods are a powerful tool for cost and time effective regulatory programs. Many microbiological inhibition and immunoassay test systems fall into this category. Results on a given sample may not be as definitive as Type I and Π methods without corroborating information. These methods may, for example, provide reasonably good quantitative information but poor compound or class specificity or identity, or may provide strong or unequivocal structural identification with very little quantitative information. Type ΙΠ methods must have defined operating characteristics of reliable performance. They are useful because of their convenience and potential suitability to non-laboratory environments, analytical speed, sample efficiency through batch analysis, portability to differing environments, sensitivity, and the ability to detect classes of compounds. A property of Type ΠΙ methods is that regulatory action based on positive results requires verification using Type I or Π methods based on the uncertainty of the Type ΙΠ individual result. However, epidemiological information may provide substantive data reducing the uncertainty of individual Type ΙΠ results. The applicability of Type ΙΠ methods should be measured, in part, by their performance characteristics, their ability to process relatively large numbers of samples within a given time frame, low use of organic solvents and their robust nature. This latter characteristic encourages the use of these qualitative and semi-quantitative methods in non-laboratory surroundings where tests may often be performed by individuals not experienced in analytical chemistry techniques. However, methods performed in nonlaboratory surroundings place constraints and needs on certain types of methodology. It a) limits use of certain types of equipment, instruments, and reagents; b) requires methods to be written in simple, unambiguous instructions that will enable a tester to


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correctly prepare the test material, conduct the analysis, interpret and report test findings; and c) requires developing simple, rugged process controls defining critical steps in die test procedure to enhance their performance and reliability. These are critical because a common response to an adverse regulatory action is to challenge the test procedure and analytical result.


Performance Characteristics To ensure analytical reliability for regulatory programs, performance characteristics ought to be determined by multi-laboratory evaluation for those methods primarily intended for laboratory use, and multi-analyst studies in non-laboratory settings for methods designed for non-laboratory uses. Minimum standards should fit the needs of specific program requirements. The principal attributes considered relevant for all types of analytical methods are its specificity, precision, systematic error, accuracy, and sensitivity. The sensitivity desired in a method is its ability to discriminate between small differences in analyte concentration. Specificity is the response of a method to the substance being measured. This characteristic is often a function of the measuring principle used or analyte functionality — key factors for rapid test methods. Methods should be able to qualitatively differentiate the analyte from analogues or metabolic products of the compound(s) of interest under the experimental conditions or use employed. Precision is the closeness of agreement between independent test results obtained from repeated measurements using separate portions of homogeneous test material under the stipulated conditions of use. It may be applied to conditions of repeatability (the same method on identical test material in the same laboratory by the same analyst using the same equipment during short time intervals) or reproducibility (the same method on identical test material in different laboratories with different analysts using different equipment). Precision is usually expressed as a standard deviation. A useful term in a regulatory program is the relative standard deviation, or coefficient of variation, because it is generally constant over a considerable concentration range (an order of magnitude, for example), ideally covering the concentration of interest. It may be reported as a percentage by dividing the standard deviation by the absolute value of the arithmetic mean and multiplying by 100. Precision is sometimes used, particularly in the European Union, to describe other method characteristics such as limit of detection and Hmit of determination. Using an analysis of a minimum of 20 blank samples, the limit of detection is expressed as the mean value for the blank determinations plus three times the standard deviation. The limit of determination is the lowest analyte content for which the method has been validated with specified degrees of accuracy and repeatability. It is commonly calculated as the mean value for the blank determinations plus six times the standard deviation (5). Precision limits agreed upon for analytical methods within the Codex Committee on Residues of Veterinary Drugs in Food, as a function of concentration, are presented in Table I (2). Within the Food Safety and Inspection Service, comparable values are used. The values listed take into consideration the wide variety of methods, analytes, matrices, and species and are usually applied in consideration of a broad-based residue control program.


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Table L Precision Guidelines for Analytical Methods.

Concentration


< 1 μg/kg >^g/kg l(^g/kg 100 μg/kg

Coefficient of Variation (Repeatability, %) 35 30 20 15

Systematic error (analytical method bias) is the difference of the measured value from the true, assigned or accepted value (mean value). It is often expressed as the percent recovery of added analyte to a sample blank. At relatively high concentrations, recoveries are expected to approach one hundred percent. At lower concentrations, and particularly with methods involving a number of steps, recoveries may be lower but need to have low variability. Accuracy refers to the closeness of agreement between the true value and the measured result. The accuracy requirements of different types of methods will vary with the use being made of the results. Accuracy requirements will vary with the objective of the test procedure. In general, methods should have their greatest accuracy at the regulatory residue limit. Hie accuracy requirement of confirmatory methods may not be as great as is required for quantification methods because in most instances these methods are only performed after a residue concentration greater than the regulatory limit has been determined by a quantification method. Suggested accuracy requirement for methods in the Codex Committee on Residues of Veterinary Drugs in Food (2) are given in Table Π, and are based upon the previously stated considerations of a broadbased residue control testing program.

Table Π. Accuracy Guidelines for Analytical Methods. Concentration < 1 Hg/kg >^g/kg 10μg/kg

Rapid Test Methods for Regulatory Programs - ACS Symposium

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