Validation requirements of the method

B. Deliverables of the Method Development Process to Support Validation Activities VALIDATION REQUIREMENTS OF THE METHOD... 

The principles of validation of residue methods for food, water and soil are generally the same. However, not all procedures and requirements are identical. From the public s point of view, the information on residues in food is probably the most important task. Compared with the other two areas (water and soil), the food sector is characterized by the largest number of regulations and legal limits. Therefore, this overview of validation requirements of enforcement methods will focus on methods for pesticide residues in food. 

Shabir, G. A. Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the U S Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. 

Abundant analytical tools exist which possess the sensitivity and a potentiality for the accuracy needed for quantitative work at the trace level in water systems. Applying these methods requires great care if the results are to be valid. Most of the methods which exist are, in their present state, limited to relatively ideal and artificial systems. They are potentially applicable to real systems, even those of considerable complexity, but the workers who use them must have skill, imagination, patience, and a feel for the statistical nature of experimental data. 

Method validation should not be considered as an isolated event. Method validation essentially begins once we develop or select a method for use. The first question to be answered is whether the method has been suitably validated by another analyst or laboratory as fit for purpose. As discussed earlier, the fitness-for-purpose statement used for this determination should include identification of the analytes and matrices to which the method is to be applied, legal limits or other concentrations that define the concentration range required of the method, plus any other considerations such as required accuracy, precision, or measurement uncertainty (MU). When suitable documentation of such validation work is available, then the method may be used by other analysts or laboratories once they have demonstrated that they can routinely attain the performance standards established in the initial validation. If evidence of such validation is not available, then experiments should be conducted to validate the method before implementation. 

However, it is the duty of the laboratory to validate each of the methods applied for drinking water analyses and to demonstrate the capability of the laboratory to fulfill the required characteristics according to the EU directive (Council Directive 98/83/EC, 1998). 

We have said that every time the calibration analyzes a new unknown sample, this amounts to an additional validation test of the calibration. It can be a major mistake to believe that, just because a calibration worked well when it was being developed, it will continue to produce reliable results from that point on. When we discussed the requirements for a training set, we said that collection of samples in the training set must, as a group, be representative in all ways of the unknowns that will be analyzed by the calibration. If this condition is not met, then the calibration is invalid and cannot be expected to produce reliable results. Any change in the process, the instrument, or the measurement procedure which introduces changes into the data measured on an unknown will violate this condition and invalidate the method If this occurs, the concentration values that the calibration predicts for unknown samples are completely unreliable We must therefore have a plan and procedures in place that will insure that we are alerted if such a condition should arise. 

Theoretically IQC should be the front-line approach to quality. If a method has been adequately validated and shown to meet the requirements of the user and kept in analytical control with IQC to detect intrusion of bias or imprecision, then the EQA needs to provide the occasional, independent, objective reassurance. In practice however, the EQA is likely to play an equal role with IQC, both in confirming problems brought to the attention of the analyst by the IQC and in stimulating further action. 

The prerequisite that the laboratory chosen to conduct the ILV trials must not be involved in the method development and/or in its subsequent use is not applicable for multi-methods. If the applicability of a multi-method is published in an official manual, an ILV is not obligatory for this particular a.i. ILV is always required for single methods. Communications between the chosen laboratory and the method developers must be reported, provided that these communications were required to carry out the analysis successfully. Also, any subsequent amendments or modifications to the original method must be reported. Furthermore, the ILV report must contain a statement as to the applicability of the method. In contrast, it is not necessary to confirm fhe resulfs of fhe enforcement methods for soil, water, body fluids, tissues, and air by an independent laboratory validation. 

The method trial process for NADA methods is different to the process for non-NADA methods. However, the validation protocol followed by the participating laboratories and the requirements for acceptance of the method are the same. The trial process also differs for determinative procedures and confirmatory procedures. Determinative procedures are evaluated using the multiple laboratory process, whereas the confirmatory method needs to be evaluated only in a single government laboratory. 

Until 1991, manufacturers seeking authorizations for pesticides had to fulfil country-specific requirements of validation of enforcement methods. The term enforcement method means analytical methods which are developed for post-registration control and monitoring purposes. The harmonization of these requirements was initiated with the European Economic Community (EEC) Council Directive 91/414/EEC and temporarily finalized with the Guidance Document on Residue Analytical Methods SANCO/825/00 rev. 6, dated 20 June 2000 [Santd et Protection des Consommateurs (SANCO)]. The evaluation of validation studies by the competent authority is conducted by comparison of these European Union (EU) requirements with the study results and most often without any practical experience of the method. Some details of this evaluation are discussed below. 

To demonstrate the validity of an analytical method, data regarding working range/ calibration, recovery, repeatability, specificity and LOQ have to be provided for each relevant sample matrix. Most often these data have to be collected from several studies, e.g., from several validation reports of the developer of the method, the independent laboratory validation or the confirmatory method trials. If the intended use of a pesticide is not restricted to one matrix type and if residues are transferred via feedstuffs to animals and finally to foodstuffs of animal origin, up to 30 sets of the quality parameters described above are necessary for each analyte of the residue definition. Table 2 can be used as a checklist to monitor the completeness of required data. 

If analytical methods are validated in inter-laboratory validation studies, documentation should follow the requirements of the harmonized protocol of lUPAC. " However, multi-matrix/multi-residue methods are applicable to hundreds of pesticides in dozens of commodities and have to be validated at several concentration levels. Any complete documentation of validation results is impossible in that case. Some performance characteristics, e.g., the specificity of analyte detection, an appropriate calibration range and sufficient detection sensitivity, are prerequisites for the determination of acceptable trueness and precision and their publication is less important. The LOD and LOQ depend on special instmmentation, analysts involved, time, batches of chemicals, etc., and cannot easily be reproduced. Therefore, these characteristics are less important. A practical, frequently applied alternative is the publication only of trueness (most often in terms of recovery) and precision for each analyte at each level. No consensus seems to exist as to whether these analyte-parameter sets should be documented, e.g., separately for each commodity or accumulated for all experiments done with the same analyte. In the latter case, the applicability of methods with regard to commodities can be documented in separate tables without performance characteristics. 

This selected ion monitoring (SIM) approach typically has greater applicability in cases where sensitivity is more of a concern. Kiehl and Kennington developed a swine liver confirmatory method for tilmicosin that confirmed structure based upon monitoring a parent ion and two additional structural fragment ions. A discussion of the validation requirements for confirmatory methods is provided in Section 6. 

Several considerations influence the suitability of the immunoassay as a qualitative or quantitative tool for the determination of tissue residues. These include the assay format, the end user (on-farm or laboratory use), effects of sample matrix on the analysis, cross-reactivity considerations, detection levels required of the assay, target tissues to be used in the assay, and the use of incurred or fortified tissues for validation of the immunoassay against accepted instrumental methods. Although these variables are often interrelated, each topic will be discussed in further detail below. 

With respect to method application, once validation has been satisfactorily completed, there is little question that use of the analytical method in worker safety and re-entry studies falls under the full requirements of the GLP Standards. In addition, there should be an adequate level of quality control measurements taken in conjunction with the specimens so as to provide for a meaningful assessment of accuracy and precision, as well as verification of freedom from artifactual interferences. Along with these measurements there needs to be reasonably rigid data acceptance criteria in place (usually established during validation) which are consistently applied during the course of the specimen analytical phase of the study. 

According to USP 28 [1], validation of an analytical method is the process by which it is established, through the conduct of laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications. Therefore, validation is an important step in determining the reliability and reproducibility of the method because it is able to confirm that the intended method is suitable to be conducted on a particular system. 

When using fluorophores of known lifetime, it is important to validate the lifetime used. Fluorescence lifetimes can be sensitive to concentration, temperature, pH, and other environmental variables. Fluorophores from different suppliers can have variable purity. As a result, one should not assume that a value reported in the literature will be exactly transferable to other labs and conditions. Users of the method should be particularly careful to use low concentrations of fluorophore (<10 /iM) to avoid a variety of processes which can perturb lifetimes in solution. There are a limited number of well characterized fluorophores. If one is not available for a particular wavelength this will require a change of filters leaving the method with nothing to recommend it over reflection and scatter. 

When no validation data are available, then all of the relevant parameters will have to be studied. The degree of rigour with which the study is carried out will depend on issues such as criticality of the measurement and the availability of validation data on similar methods. There will be cases in the laboratory where a method has been used, satisfactorily, for a long period of time but there is no documentation to demonstrate the performance of the method. It seems unreasonable to require full revalidation when a method has been used successfully for some years. However, the need for objective evidence prevents the validity of such a method being taken for granted. A possible approach is to follow the plan below ... 

Identify significant issues that require further attention and provide the missing information. Unless the validity of the method is in serious doubt, the method can continue to be used. However, the new validation information should be produced over a reasonable time-period and may involve experimental data and professional judgement. 

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