Analytical method validation defined

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, 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. 

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. 

Method validation must be performed in a regulatory-compliant environment. In particular, the organization must have a QA unit (QAU), adequate laboratory equipment and facilities, written procedures, and qualified personnel. Since a successful validation requires the cooperative efforts of each of these organizational elements, successful fulfillment of the regulatory and technical objectives of validation requires senior management support. Additionally, it is essential that the organization have a well-defined validation master plan (YMP) for analytical methods, which defines the steps necessary to effectively validate methods. 

An important fact inherent in the purity analysis of a recombinant pharmaceutical is that the absolute purity of any protein is an elusive, if not an unobtainable, measurement. For biopharmaceuticals, purity is a relative term. Protein purity is method-dependent and is defined by the shortcomings of the analytical procedure. Also, unlike small traditional drugs, proteins are highly complex molecules. For these two reasons, more than one method must be utilized to define a protein s purity. The greater the number of methods used in the purity analysis, the greater the assurance is that the product is pure. Furthermore, the purity determined by an analytical method can only be properly interpreted based on the method s validation. Analytical methods validation is critical to and inseparable from purity determinations. A detailed discussion on analytical methods validation is beyond the scope of this chapter but other sources of information are available for the interested reader.11 13... 

To the pharmaceutical world, the meaning of analytical methods validation is the process to confirm that a method does what it purports to do, that is, to document through laboratory studies that the measurement procedure can reliably assess the identity, strength, and/or quality of a bulk drug substance, excipient, or finished pharmaceutical product. To provide consistent, worldwide regulatory expectations, previously unavailable, the International Conference on Harmonization has defined the methods validation... 

An attempt for harmonization was made at the International Conference on Harmonization (ICH) in 1995 and 1996 by representatives from the industry and regulatory agencies from the United States, Europe, and Japan, who defined parameters, requirements, and, to some extent, also methodology, for analytical methods validation. 

Process validation, formally articulated in 1987 and similar in theory to analytical method validation, is defined as ... establishing documented evidence which provides a high degree of assurance that a specihc process will consistently produce a product meeting its pre-determined specihcations and quality attributes. ... 

Analytical methods and specifications must be established and validated so as to define and control the quality and purity of the raw materials, intermediates and the finished product. For many standard chemical raw materials, the development of specifications will not be necessary as they are already published in US and European pharmacopoeia (for example, standards for water, organic solvents and various excipients). The ultimate objective of these activities is to be able to manufacture the drugs required for clinical trials in accordance with good manufacturing practice (GMP). 

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. 

In a widely accepted definition, an analytical method can be defined as the series of procedures from receipt of a sample to the production of the final result. Often, not all procedures can be validated in an adequate way. However, even in such cases, where all procedures of a method are validated, the performance characteristics obtained do not reflect all sources of error. In a recent paper,the complete ladder of errors is described in the following way ... 

But in order to understand what needs to be changed, we first need to understand the current situation. In order for a pharmaceutical company to use any analytical method for certifying the properties (efficacy, potency, etc.) of their products, the analytical method has to be validated. Validation , in the parlance of the FDA, is a far cry from what we usually call validation when developing a multivariate spectroscopic method. In fact, what we call validation in spectroscopic calibration (which usually means calculating an SEP, or an SECV) is a far cry from the dictionary definition of validate , which is to make legally valid , where valid is defined as having legal efficacy or force [11],... 

Participants can use the analytical method of their choice except when otherwise instructed to adopt a specified method. It is recommended that all methods should be properly validated before use. In situations where the analytical result is method-dependent the true value will be assessed using those results obtained using a defined procedure. If participants use a method that is not equivalent to the defining method, then an automatic bias in result will occur when their performance is assessed. 

There are occasions where new analytical methods have to be developed specifically for testing raw materials, intermediates, and finished products that are not covered by compendial methods. In these situations, the analytical methods are required to undergo a validation process to ensure they are suitable. One or more of the following parameters as defined in Exhibit 9.10 must be validated for newly developed analytical methods ... 

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... 

Before a calibration is started, the lowest and highest concentration, i.e., the concentration range of interest, must be defined. Then the method must be validated over the entire range. The range of an analytical method is the interval between the upper and... 

Finally, process analytics methods can be used in commercial manufacturing, either as temporary methods for gaining process information or troubleshooting, or as permanent installations for process monitoring and control. The scope of these applications is often more narrowly defined than those in development scenarios. It will be most relevant for manufacturing operations to maintain process robustness and/or reduce variability. Whereas the scientific scope is typically much more limited in permanent installations in production, the practical implementation aspects are typically much more complex than in an R D environment. The elements of safety, convenience, reliability, validation and maintenance are of equal importance for the success of the application in a permanent installation. Some typical attributes of process analytics applications and how they are applied differently in R D and manufacturing are listed in Table 2.1. 

Validation of analytical methods is important both from the standpoint of good science and to satisfy regulators of the reliability of results reported in dossiers. When approaching validation of any analytical method, one must always ensure that the method is fit for purpose. The amount of resource should be appropriate to the phase of development and the degree to which the process is defined and the methods finalised. 

The quality plan defines the inputs and outputs of any laboratory process. For example, the quality plans refer to the analytical methods, the irrstnrments and laboratory equipment that are used for the analysis, the characteristics of the analysis results etc. All laboratory processes (e g. analytical methods) and laboratory products (e g. analytical resrrlts) should be subject to verification and validation to ensme that they are fit for the ptrrpose. The acceptance criteria should be defined and records should always be kept as evidence of meeting the requirements. 

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]. 

Extent of Validation Depends on Type of Method On the one hand, the extent of validation and the choice of performance parameters to be evaluated depend on the status and experience of the analytical method. On the other hand, the validation plan is determined by the analytical requirement(s) as defined on the basis of customer needs or as laid down in regulations. When the method has been fully validated according to an international protocol [63,68] before, the laboratory does not need to conduct extensive in-house validation studies. It must only verify that it can achieve the same performance characteristics as outlined in the collaborative study. As a minimum, precision, bias, linearity, and ruggedness studies should be undertaken. Similar limited vahdation is required in cases where it concerns a fully validated method which is apphed to a new matrix, a well-established but noncol-laboratively studied method, and a scientifically pubhshed method with characteristics given. More profound validation is needed for methods pubhshed as such in the literature, without any characteristic given, and for methods developed in-house [84]. 

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. 

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