Single laboratory method validation

B. Richter, J. EzzeU, and D. Felix, Single Laboratory Method Validation Report Extraction of Organo-phosphorus Agrochemicals, Chlorinated Herbicides and Polychlorinated Biphenyls Using Accelerated Solvent Extraction (ASE) with Analytical Validation by GC/NPD and GC/ECD, Document 101124, Dionex Sunnyvale, CA (1994). 

As stated by IUPAC [5], a validation method can be differentiated into categories of fully validated and single-laboratory method validation. A fully validated... 

There is concern in the food analytical community that although methods should ideally be validated by a collaborative trial, this is not always feasible for economic or practical reasons. As a result, IUPAC guidelines are being developed for single laboratory method validation to give information to analysts on the acceptable procedure in this area. These guidelines should be finalised by the end of 2001. 

There are two approaches to single-laboratory method validation The traditional one that identifies and then evaluates the set of analytical parameters, and a more recent one that is based on the evaluation of uncertainty. 

The ISO 17025 requires that every method is validated to demonstrate fitness for purpose [1]. Analytical methods can be validated during a collaborative trial or in a single laboratory validation experiment. Single laboratory method validation has become common practice for every analytical chemist. Several guidance documents have been published concerning the validation of analytical methods [2, 3]. Methods used for the official control of foodstuffs are validated according to the European decision EC 2002/657 [4]. 

There are two sources of information from the CAC that provide guidance on the validation of analytical methods. General guidance on method validation, including single-laboratory method validation, may be found in the CAC Procedural Manual.This is supplemented by Codex Alimentarius Commission guidelines, a number of which have been adopted from harmonized guidelines previously developed by independent international scientific... 

Precision, which quantifies the variation between replicated measurements on test portions from the same sample material, is also an important consideration in determining when a residue in a sample should be considered to exceed a MRL or other regulatory action limit. Precision of a method is usually expressed in terms of the within-laboratory variation (repeatability) and the between-laboratory variability (reproducibility) when the method has been subjected to a multi-laboratory trial. For a single-laboratory method validation, precision should be determined from experiments conducted on different days, using a minimum of six different tissue pools, different reagent batches, preferably different equipment, and so on, and preferably by different analysts Repeatability of results when determined within a single laboratory but based on results from multiple analysts is termed intermediate precision Precision of a method is usually expressed as the standard deviation. Another useful term is relative standard deviation, or coefficient of variation (the standard deviation divided by the absolute value of the arithmetic mean result, multiplied by 100 and expressed as a percentage). 

Two types of method validation can be distinguished. Full method validation, of interest to the general scientific community, is carried out through an interlaboratory method performance study. Where a method becomes more routinely used it is reasonable to expect that the method should be fully validated. Internal method validation (single-laboratory method validation) is a scientific and technical alternative. It consists of validation steps carried out within one laboratory, for instance, to validate a new method that has been developed in-house or to verify that a method adopted from some other source is applied sufficiently well. A single-laboratory validation cannot assess between-laboratory variation and will provide an optimistic assessment of interlaboratory variability (cfr. Chp. 6.2.3 and 6.2.4). In-house method validation is described in the lUPAC, AOAC International, and ISO guidance [65,66]. There are several types of internal laboratory validation ... 

Each individual method collection comprises a large number of methods, which often have different validation statuses. For instance, the most important Swedish multi-residue method (based on ethyl acetate extraction, GPC and GC) is validated for many pesticides by four laboratories, but other methods are presented with singlelaboratory validation data. Some methods in the Dutch and German manuals were tested in inter-laboratory method validation studies, but others by an independent laboratory or in a single laboratory only. 

EPA. 1987d. Project summary Single laboratory validation of EPA method 8140. Las Vegas, NV U.S. Environmental Protection Agency. EPA 600/S4-87-009. 

Non-NADA methods may be designed to detect multiple residues and they may be designed for use in multiple species. In order to validate these multi-residue methods, modifications to the validation protocol relative to single analyte methods are made. Additional laboratories will participate in the method trial, but the number of samples... 

Table 8 Summary of CSL parameters and criteria for single-laboratory validation of procedures involved in a quantitative method ... 

Because of the small number of laboratories involved, validation of UK methods by inter-laboratory study has become impractical in most cases. Even where it is practical, it is usually impossible to validate all pesticide-matrix combinations. Moreover, single-laboratory validation data will have to be generated. Therefore, the CSL guidelines are one of the first that strictly focus on requirements of single-laboratory validation. Some examples of minimum requirements are given in Table 8. Additionally, these guidelines emphasize some other important aspects of validation and contain some new ideas. 

Even if most examples and procedures presented apply to in-house validation, the procedure does not distinguish between validations conducted in a single laboratory and those carried out within inter-laboratory method performance studies. A preference for inter-laboratory studies can be concluded from the statement that laboratories should always give priority to methods which have been tested in method performance studies. Within the procedure a profound overview of different categories of analytical methods according to the available documentation and previous external validation is given. For example, if a method is externally validated in a method performance study, it should be tested for trueness and precision only. On the other hand, a full validation is recommended for those methods which are published in the scientific literature without complete presentation of essential performance characteristics (Table 9). 

The workhorses in national monitoring programs are multi-residue methods. Any official method collection of any EU Member State contains at least one multi-residue method. For multi-analyte and/or multi-matrix methods, it is likely to be impractical to validate a method for all possible combinations of analyte, concentration and type of sample matrix that may be encountered in subsequent use of the method. Therefore, initial validation should incorporate as many of the target analytes and matrices as practicable. For practical reasons this validation and the evaluation of other methods with limited scope often cannot be conducted in inter-laboratory studies. Other concepts based on independent laboratory validation or validation in a single laboratory have been developed and can provide a practical and cost-effective alternative (or intermediate) approach. 

The magnitude of these errors can be analyzed in single laboratories (run bias and repeatability error), in inter-laboratory validation studies (laboratory bias) and in proficiency tests (method bias). Expressed in standard deviations relative to that of... 

AOAC/FAO/IAEA/IUPAC Expert Consultation, Guidelines for Single Laboratory Validation of Analytical Methods for Trace-level Concentrations of Organic Chemicals, Workshop, 8-11 November 1999, Miskolc, Hungary (1999). Also available on the Word Wide Web http //www.iaea.oi trc/(see pesticides —> method validation). 

Horwitz points out the universal recognition of irreproducible differences in supposedly identical method results between laboratories. It has even been determined that when the same analyst is moved between laboratories that the variability of results obtained by that analyst increases. One government laboratory study concluded that variability in results could be minimized only if one was to conduct all analyses in a single laboratory. .. by the same analyst . So if we must always have interlaboratory variability how much allowance in results should be regarded as valid - or legally permissible as indicating identical results. What are the practical limits of acceptable variability between methods of analysis - especially for regulatory purposes. 

An indication of the minimum size of a subsample can be obtained by using the concept of a sampling constant. For example, in the laboratory, the sampling constant can be used to estimate the minimum size of the test portion. However, the suitability of the chosen test portion size must be confirmed as part of method validation. The sampling constant Ks has units of mass. This is the mass of the test portion necessary to ensure a relative subsampling error of 1% (at the 68% confidence level) in a single determination. The value of /Ks is numerically equal to the coefficient of variation, CV (see Chapter 6, Section 6.1.3) for results obtained on 1 g subsamples in a procedure with insignificant analytical error. 

If the analytical method used by participants in the proficiency testing round has been validated by means of a formal collaborative trial, then the repeatability and reproducibility data from the trial can be used. The repeatability standard deviation gives an estimate of the expected variation in replicate results obtained in a single laboratory over a short period of time (with each result produced by the same analyst). The reproducibility standard deviation gives an estimate of the expected variation in replicate results obtained in different laboratories (see Chapter 4, Section 4.3.3 for further explanation of these terms). 

Use of Validated Methods In-Home Versus Interlaboratory Validation Wherever possible or practically achievable, a laboratory should use methods which have been fully validated through a collaborative trial, also called interlaboratory study or method performance study. Validation in collaborative studies is required for any new analytical method before it can be published as a standard method (see below). However, single-laboratory validation is a valuable source of data usable to demonstrate the fitness for purpose of an analytical method. In-house validation is of particular interest in cases where it is inconvenient or impossible for a laboratory to enter into or to organize itself a collaborative study [4,5]. 

On the one hand, even if an in-house vahdated method shows good performance and reliable accuracy, such a method cannot be adopted as a standard method. In-house validated methods need to be compared between at least eight laboratories in a collaborative trial. On the other hand, a collaborative study should not be conducted with an unoptimized method [58]. Interlaboratory studies are restricted to precision and trueness while other important performance characteristics such as specificity and LOD are not addressed [105]. For these reasons, single-laboratory validation and interlaboratory validation studies do not exclude each other but must be seen as two necessary and complementary stages in a process, presented in Figure... 

The added value of single-laboratory validation is that it simplifies the next step—interlaboratory validation—and thereby minimizes the gap between internally (validated or not) developed methods and the status of interlaboratory validation. By optimizing the method first within the laboratory, as a kind of preliminary work, an enormous amount of collaborators time and money is saved [58]. 

The foregoing discussion is encapsulated in figure 8.1, which shows that method validation can be seen as checking and signing off on a method development cycle, with the important caveat that initial development is often done in a single laboratory and so will not usually establish interlaboratory... 

How these different effects are treated depends on the nature of the method validation. For the validation of a new method by an international body, including an extensive interlaboratory study, the method bias and reproducibility will be reported. For a single laboratory, laboratory bias and intermediate reproducibility will be determined. 

Thompson, M, Ellison, S, and Wood, R (2002), Harmonized guidelines for single laboratory validation of methods of analysis. Pure and Applied Chemistry, 74, 835-55. 

Cross-Validation. Cross-validation is a comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same study or across different studies. An example of cross-validation would be a situation where an original validated bioanalytical method serves as the reference and the revised bioanalytical method is the comparator. The comparisons should be done both ways. When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted at each site or laboratory... 

When the method development process has been completed, an analytical procedure is subject to validation and transfer to routine use. This process may be called technology transfer which implies migration from the R D environment to routine analytical laboratories. This process needs to be carried out whether the applicability of the procedure is limited to a single laboratory or to many laboratories. 

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