Validation Requirements for Testing for Residues of Veterinary Drugs

James D. MacNeil, Saskatoon, Saskatchewan, Canada

Background

Codex guidelines for the assessment of the competence of testing laboratories involved in the import and export control of foods (CAC/GL 27^[55]) specify that such laboratories should adopt quality criteria which include compliance with the general criteria found in ISO/IEC Guide 25^[56] (since updated and replaced by ISO/IEC-17025^[57]), participate in appropriate proficiency schemes conducted according to international guidelines^[58], use internal quality control procedures such as described in an international guideline^[59] and "when available, use methods of analysis which have been validated according to the principles laid down by the Codex Alimentarius Commission".

General guidance on validation requirements for methods may be found in the Procedural Manual^[60]. These are used specifically by the Codex Committee on Methods of Analysis and Sampling to identify methods suitable for use to support a standard established by a Codex Commodity Committee. While methods for analysis of residues of veterinary drug residues in foods are excluded from this process, the technical criteria established by the Committee on Residues of Veterinary Drugs in Foods are similar to those elaborated by CCMAS and included in the Procedural Manual. Ideally, a method recommended to support a Codex standard, particularly one designated as a Codex reference method, will have been the subject of a full collaborative study^[61] to establish the performance criteria which should be achieved by an analyst using the method. In practice, such studies have rarely been completed for methods intended for analysis of veterinary drug residues in foods. Collaborative studies completed during the past decade included liquid chromatographic methods for sulfonamides^[62] and tetracyclines^[63] in bovine milk, tetracyclines in animal tissues^[64] and clopidol in chicken muscle^[65]. Factors contributing to this relatively small number of studies include the limited number of expert laboratories which can devote time to the work within the project schedule, difficulties in generating incurred residues at the required concentrations, sample stability and the complex issues associated with international shipment of biological materials. Such studies are expensive, time-consuming and may be overtaken by changes in regulation or technology.

Method Validation Relationship to Maximum Residue Limits (CCRVDF)

At the 9th Session of the Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF), it was agreed that Maximum Residue Limits (MRL s) "should be developed independently of validated methods, but such methods should be available before the Committee advances MRLs to Step 8"^[66]. However, it became apparent at subsequent meetings of the Committee that methods validated by full collaborative study were not available to support most MRL recommendations. The Codex Alimentarius, Volume 3, Residues of Veterinary Drugs in Foods, 2nd edition, defines a validated method as "an analytical method which has been subjected to a multi-laboratory study for accuracy, precision, reproducibility performance and ruggedness"^[67]. In practice, the CCRVDF required that information should be provided to demonstrate method validation by a minimum of three analysts, preferably working independently in three laboratories. While this provided a number of methods that could be recommended by the Committee, it was found that the changing laboratory environment, with an increasing focus on accreditation and demonstration of "fitness for purpose" of a method by laboratories during accreditation meant that there was less interest in multi-laboratory trials of their methods by regulatory laboratories. Instead, laboratories relied more on internal quality systems and participation in proficiency sample exchanges to demonstrate "method validation".

The Criteria Approach and Single Laboratory Method Validation

A result of the "criteria approach" to method validation, which is now found in general terms in the Procedural Manual and in more specific detail in Commission Decision 2002/657/EC concerning the performance of analytical methods used in regulatory laboratories testing for veterinary drug residues in the European Union^[68], was a recognition that the reliance on a multi-laboratory method trial as the basis for method validation may no longer be appropriate. On advice from the 10th Meeting of the CCRVDF, the Codex Alimentarius Commission requested the Food & Agriculture Organization convene an expert consultation on "methods validation for food control purposes"^[69]. The resulting Consultation, held in Vienna in 1997^[70], recommended that "all methods used for determining compliance with international or other standards which have not been subjected to a full collaborative study should be subject to a form of independent review, which may include any of the following options: a) a multi-laboratory validation study involving a smaller number of laboratories, b) second laboratory verification, in a laboratory operating under Good Laboratory Practices (GLP) or c) validation in a laboratory which has been recognized under ISO/IEC Guide 17025 or equivalent." The Consultation recommended the following principles which should be applied in cases "where collaborative or other interlaboratory studies are impractical or impossible to carry out":

1. Laboratories carrying out the validation studies operate under a suitable quality system based upon internationally recognized principles^[59] .
   
   
2. Laboratories have in operation a third party review of the whole validation process (e.g., GLP registration, accreditation according to ISO/IEC 17025 or equivalent, or Peer Review).
   
   
3. Analytical methods are assessed in respect to the Codex general criteria for selection of methods of analysis^[60], with emphasis on the assessment of the limit of quantitation rather than the limit of detection.
   
   
4. The validation work should be carefully documented in an expert validation report in which it is unambiguously stated for which purposes (matrices and analyte concentrations) the method has been found to perform in a satisfactory manner.
   
   
5. Evidence of transferability should be provided for all methods intended for Codex use for food control purposes.

Subsequently, requirements for "single laboratory method validation" were considered by a working party of the International Union of Pure and Applied Chemistry (IUPAC), resulting in the publication of a general guidance document^[71]. An expert consultation jointly sponsored by IUPAC, AOAC International. the International Atomic Energy Agency (IAEA) and the FAO also issued a report providing specific guidance for the validation of methods intended for the determination of trace residues of pesticides and veterinary drugs in foods^[72],[73]. The contents of the IUPAC guidance document and the consultation report are being used by experts from both the Codex Committee on Pesticide Residues (CCPR) and the CCRVDF in revising requirements for the validation of analytical methods recommended by the two committees for, respectively, pesticide residues and veterinary drug residues.

What, then, is meant by method validation? Simply defined, method validation is a process using a defined set of experiments to establish the performance criteria which should be achieved by an analyst using the method and thereby to provide to both users of the method and their clients a means to assess the reliability of results obtained using the method. The necessary experiments must be conducted to establish the various performance criteria defined in the Procedural Manual and in other relevant Codex documents, such as the Codex Alimentarius, Volume 3, including analytical recovery, precision at various concentrations within the defined analytical range of the method and detection capabilities, conventionally expressed in such terms as selectivity, specificity and limits of detection and quantification. The requirements vary according to the type of method and the intended use. The requirements for validation of a method used qualitatively as a screening method differ from those for determinative or quantitative methods, while additional criteria apply to confirmatory methods. Certain elements, such as analyte and sample stability, are common to all types of methods and once determined, do not require validation when a new method is being validated unless there are significant changes which could affect the stability (for example, a significant change in a temperature or extraction conditions).

Using the criteria approach to validation and the context of an ISO-17025 laboratory accreditation, or equivalent, the validation data will reflect the current use of a method. Initially, a method may be validated in a single laboratory by a single analyst. That validation provides the most restricted form of information, as it provides a set of performance criteria that may be unique to that analyst and analytical system (reagents, instruments, laboratory environment, etc.). Such a validation may be all that can be achieved in a laboratory with limited staff and resources or when a method is first being introduced. The degree of validation is increased when the method is used by one or more additional analysts in the same laboratory, then broadened further when different sources of reagents and different instruments are used. The generation of data in one or more additional laboratories provides further confidence in the method performance, with the full collaborative study being considered to offer the most desirable level of statistical assessment of method performance criteria. In many cases, regulatory methods are used by a limited number of analysts in only one or a few laboratories, so the establishment of performance standards for those conditions of use defines the "fit for purpose" requirement. It is the responsibility of the laboratories using the method to generate the data which provide evidence of method validation and also which demonstrate the ability of each analyst to achieve the performance standards established during the validation. Inter-laboratory variability is certainly an issue when multiple laboratories may use the same method, particularly if replicate test portions of the same sample material are involved. When the method is used only in one laboratory, the performance of the analyst with that method in comparison with other analysts using different methods in other laboratories may become the issue. However, whether validation is conducted by a single analyst or by multiple analysts at multiple locations, a key element of the work which should be undertaken either prior to or during the formal validation is ruggedness testing^[74] to establish critical points in the method where a minor variation in procedure or a change in source of material or reagent can profoundly alter the test result.

New Issues and Challenges

In the case of substances for which no ADI (Acceptable Daily Intake) or MRL has been established, the issue for the regulator may be the proven presence or absence of a residue in a food rather than the quantity present. In such cases, the critical issues for validation may be the selectivity and sensitivity of the screening test and the lowest concentration at which a confirmatory test can detect and confirm the presence of residues. Experiments suitable to provide statistically-based evidence of performance of screening tests have been described as used in a performance verification program established to demonstrate the suitability of test kits^[75]. For validation of confirmatory methods based on mass spectrometry, requirements have been elaborated in documents published by the European Commission^[68] the United States Food and Drug Administration^[76] and the American Society for Mass Spectrometry^[77]. These require the presence of a match with the chromatographic retention time of a standard, the presence of a minimum number of characteristic (structurally significant) fragment ions and an agreement within specified limits between the ratios of the ions present in a standard with those present in the sample.

The introduction of Minimum Required Proficiency Limits (MRPLs) in European Commission regulations^[68] and the subsequent promulgation of MPRLs for selected compounds^[78] has introduced an additional dimension into the validation issue. The MRPLs set minimum criteria for detection and confirmation for such substances which must be met by regulatory laboratories within the European Community member states and, by extension, by official laboratories which certify products for export from other countries to the EU. It is not clear whether validation of methods used for such purposes must include quantitative application, or simply the ability to screen and confirm at the target level with a specified statistical confidence. Such validation requirements are not currently addressed within the criteria established by CCRVDF, which focus on substances for which an ADI and MRLs have been established.