Method validation defined

A reference method is defined as a method of known and proven accuracy, thoroughly validated, and experimentally demonstrated to have negligible systematic errors and a high level of precision. Its development involves removing the principal systematic errors of the process, reducing them to tolerable levels, or when actual... 

Today, when a pesticide with no detectable residues is registered for use, a Tolerance or maximum residue limit (MRL) is established at the lowest concentration level at which the method was validated. However, for risk assessment purposes it would be wrong to use this number in calculating the risk posed to humans by exposure to the pesticide from the consumption of the food product. This would be assuming that the amount of the pesticide present in all food products treated with the pesticide and for which no detectable residues were found is just less than the lowest level of method validation (LLMV). The assumption is wrong, but there is no better way of performing a risk assessment calculation unless the limit of detection (LOD) and limit of quantification (LOQ) of the method were clearly defined in a uniformly acceptable manner. 

Finally, it is important to define the lowest level of method validation (LLMV). The LLMV is defined as the lowest concentration level expressed in terms of amount of analyte in the matrix, at which the method (extraction/analysis procedure) was validated or proven to be capable of reliably quantifying. 

The method should define all criteria used to determine if an analysis is valid and the data are acceptable as part of the SOP. The analyst may not substitute or modify criteria used to determine the acceptance of data after an analysis has been completed. 

In this article, an analytical method is defined as series of procedures from receipt of a sample to final determination of the residue. Validation is the process of verifying that a method is fit for purpose. Typically, validation follows completion of the development of a method. Validated analytical data are essential for monitoring of pesticide residues and control of legal residue limits. Analysts must provide information to demonstrate that a method intended for these purposes is capable of providing adequate specificity, accuracy and precision, at relevant analyte concentrations and in all matrices analyzed. 

Method validation guidelines for use in trace analysis have been proposed by various authors, but there is little consistency in the recommended approaches. The general validation guidelines proposed by standards organizations such as ISO (International Organization for Standardization), DIN (Deutsches Institut fUr Normung German Institute for Standardization) and others are often not well defined and consequently... 

The sensitivity achieved (LOD) is not normally presented. It is recognized that different laboratories determine dissimilar values for this parameter and even within a laboratory the repeatability of the LOD is low. Most often, the lowest validated concentration gives an impression about the lowest levels that can be analyzed generally with acceptable results. A measure of selectivity is the intensity of blank results. This intensity is discussed by the participants of inter-laboratory validation studies. However, results are not reported and limits are not defined by CEN TC 275. The results of method validations of the several multi-residue/multi-matrix methods are not reported in the same way, but newer methods with limited scope generate analogous tables with validation results (as an example, see Table 7). 

RACs. The LOD and LOQ for each study should be determined. Until each study s LOD and LOQ have been determined, use the lower limit of method validation (LLMV) of 0.01 mgkg for each metabolite class as the reference LOQ. LLMV is defined as the lowest level of fortification where we have demonstrated acceptable recovery and precision of EMA- and HEMA-producing metabolites. 

Method validation, on the other hand, is normally considered part of the study in which the method will subsequently be used or consists of a separate defined study unto itself as such, it is normally required to be accomplished under GLP purview. There is, however, some confusion in some circles as to exactly what is meant by analytical method validation. Some chemists describe it as adaptation of one method from one type of matrix for use with another using basically the same or similar analytical approach. Others take a more strict interpretation and define validation as simply demonstration of the ability to achieve satisfactory results using a published procedure in one s own laboratory setting. Often, validation incorporates both interpretations. 

Method validation is defined in the international standard, ISO/IEC 17025 as, the confirmation by examination and provision of objective evidence that the particular requirements for a specific intended use are fulfilled. This means that a validated method, if used correctly, will produce results that will be suitable for the person making decisions based on them. This requires a detailed understanding of why the results are required and the quality of the result needed, i.e. its uncertainty. This is what determines the values that have to be achieved for the performance parameters. Method validation is a planned set of experiments to determine these values. The method performance parameters that are typically studied during method validation are selectivity, precision, bias, linearity working range, limit of detection, limit of quantitation, calibration and ruggedness. The validation process is illustrated in Figure 4.2. 

The context in which the localization method is defined has already been explained in the introductory Section I, but although the method itself is well known the physical basis of its premises remains in many ways obscure. In particular, the concept of localization of tt electrons requires clarification, and the validity of theoretical relationships between reactivity indices of the isolated molecule and localization methods needs further discussion. In this Section we recall the original statement of the method in some detail, and then review some subsequent developments the relationship between the two methods is discussed in Section VI. 

A validation protocol adapted from the experiences during the method development defines the scope of the validation study (goal of the study, regulating guidelines, key method parameters, etc.). To investigate the adequate method performance, these features (e.g., range of analyte concentration), together with a statement of any fitness-for-purpose criteria, have to be specified in the validation protocol. A basic check has to provide that the reasonable assumptions about the principles of the method are not seriously flawed. In this process, sources of error in analysis have to be listed (Table 4) and their effects have to be checked. The validation should, as far as possible, be conducted to provide a realistic survey of the number... 

Optimal values for the factors are selected from the tested levels for the factors (extremes or nominal) in function of a number of responses of the method (see also references [16,19]). When one changes the method conditions due to these results one has to be aware that a new method is defined. What is done here is in fact a simplistic way of optimizing a method. The optimization of a method however is a step that is expected to come much sooner in the method development than in the ruggedness testing. One also has to realize that when one defines a new method this requires a new full validation, including a ruggedness test. 

Measurement uncertainty is the most important criterium in both method validation and IQC. It is defined as a parameter, associated with the result of a measurement, that characterizes the dispersion of the values that could reasonably be attributed to the measurand [14]. The measurand refers to the particular quantity or the concentration of the analyte being measured. The parameter can be a standard deviation or the width of a confidence interval [14, 37]. This confidence interval represents the interval on the measurement scale within which the true value lies with a specified probability, given that all sources of error are taken into account [37]. Within this interval, the result is regarded as being accurate, that is, precise and true [11]. 

The ISO definition of validation is confirmation by examination and provision of objective evidence that the particular requirements of a specified intended use are fulfilled [15]. Method validation is needed to confirm the fitness for purpose of a particular analytical method, that is, to demonstrate that a defined method protocol, applicable to a specified type of test material and to a defined concentration rate of the analyte —the whole is called the analytical system — is fit for a particular analytical purpose [4]. This analytical purpose reflects the achievement of analytical results with an acceptable standard of accuracy. An analytical result must always be accompanied by an uncertainty statement, which determines the interpretation of the result (Figure 6). In other words, the interpretation and use of any measurement fully depend on the uncertainty (at a stated level of confidence) associated with it [8]. Validation is thus the tool used to demonstrate that a specific analytical method actually measures what it is intended to measure and thus is suitable for its intended purpose [11,55,56]. 

This chapter has two aims to demonstrate the necessity of using properly validated and verified methods and to explain what constitutes a validated method, and to provide an introduction to method validation for in-house methods. There is an abundance of published material that defines, describes, and generally assists with method validation, some of which is... 

Limit of detection (LOD) sounds like a term that is easily defined and measured. It presumably is the smallest concentration of analyte that can be determined to be actually present, even if the quantification has large uncertainty. The problem is the need to balance false positives (concluding the analyte is present, when it is not) and false negatives (concluding the analyte is absent, when it is really present). The International Union of Pure and Applied Chemistry (IUPAC) and ISO both shy away from the words limit of detection, arguing that this term implies a clearly defined cutoff above which the analyte is measured and below which it is not. The IUPAC and ISO prefer minimum detectable (true) value and minimum detectable value of the net state variable, which in analytical chemistry would become minimum detectable net concentration. Note that the LOD will depend on the matrix and therefore must be validated for any matrices likely to be encountered in the use of the method. These will, of course, be described in the method validation document. 

Method Procedure. Since the method procedure is undergoing constant modifications during method development, it is very important to define the procedure before method validation. This will ensure that the same method procedure will be used in all method validation experiments. 

System Suitability Tests. The appropriate system suitability tests should be defined before method validation (e.g., precision, resolution of critical related substances, tailing, detector sensitivity). These system suitability tests should be performed in each method validation experiments. System suitability results from the method validation experiment can be used to determine the appropriate system suitability acceptance criteria. 

The validation requirements are discussed as they apply to both the sample preparation and sample analysis aspects of a dissolution method. The focus of the discussion in this chapter is on the validation considerations that are unique to a dissolution method. Validation is the assessment of the performance of a defined test method. The result of any successful validation exercise is a comprehensive set of data that will support the suitability of the test method for its intended use. To this end, execution of a validation exercise without a clearly defined plan can lead to many difficulties, including an incomplete or flawed set of validation data. Planning for the validation exercise must include the following determination of what performance characteristics to assess (i.e., strategy), how to assess each characteristic (i.e., experimental), and what minimum standard of performance is expected (i.e., criteria). The preparation of a validation protocol is highly recommended to clearly define the experiments and associated criteria. Validation of a test method must include experiments to assess both the sample preparation (i.e., sample dissolution) and the sample analysis. ICH Q2A [1] provides guidance for the validation characteristics of the dissolution test and is summarized in Table 4.1. 

During the 1990 Washington Conference on Analytical Methods Validation Bioavailability, Bioequivalence and Pharmacokinetic Studies [1], parameters that should be used for method validation were defined. The final report of this conference is considered the most comprehensive document on the validation of bioanalytical methods. Many multinational pharmaceutical companies and contract research organizations contributed to its final draft. This scientific meeting was sponsored by the American Association of Pharmaceutical Scientists (AAPS), the Association of Official Analytical Chemists (AOAC), and the U.S. Food and Drug Administration (FDA). The conference report has been used as a reference by bioanalytical laboratories and regulatory agencies worldwide. 

System Suitability. Although method validation is performed once at the end of method development, system suitability tests are performed on a specific system periodically (usually daily) or prior to each batch during validation and sample analysis to determine the system performance (see Chapter 13). During method development or/and upon completion of the validation, system suitability data should be evaluated and used to define acceptance criteria to use before starting sample analysis. System suitability tests include (1) the reproducibility of retention time, (2) adequate sensitivity to quantify LLOQ (minimum detector response), (3) appropriate sensitivity to quantify ULOQ (within range of detector), and (4) chromatographic separation. 

During method validation the parameters, acceptance limits, and frequency of ongoing system suitability tests or quality control checks should be defined. Criteria should be defined to indicate when the method and system are out of statistical control. The goal is to optimize these experiments in such a way that with a minimum number of control analyses the method and the complete analytical system will provide long-term results that will meet the objectives defined in the scope of the method. 

The phrase related to implies that the relationship is known and valid. This will only be realized if the relationship at every step of the process is clearly defined and valid. Hence the requirement for an unbroken chain of comparisons. The parallel between these issues and those addressed by method validation is worth noting. Validation is the process of establishing that a method is capable of measuring the desired measurand (analyte), with appropriate performance characteristics, such as level of uncertainty, robustness, etc. It should also address systematic effects, such as incomplete recovery of the analyte, interferences, etc. These latter issues can be dealt with by designing a method to eliminate any bias, at a given level of uncertainty, or if that is not possible, to provide a means of correcting for the bias. This may be done at the method level, by applying a correction factor to all results, or at the individual measurement level. 

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