Validation Collaborative trial

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). 

Consideration of the above requirements confirms that in future all methods must be fully validated if at all possible, i.e. have been subjected to a collaborative trial conforming to an internationally recognised protocol. In addition this, as described above, is now a legislative requirement in the food sector of the European Union. The concept of the valid analytical method in the food sector, and its requirements, is described below. 

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. 

Has the method been validated by collaborative trial (i.e. externally) ... 

Validation of a new analytical method is typically done at two levels. The first is the level of prevalidation, aiming at fixing the scope of the validation. The second level is an extensive, full validation performed through a collaborative trial or interlaboratory study. The objective of full validation, involving a minimum number of laboratories, is to demonstrate that the method performs as was stated after the prevalidation. 

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... 

Precision plays a central role in collaborative studies. Wood [84] defines a collaborative trial as a procedure whereby the precision of a method of analysis may be assessed and quantified. Precision is the objective of interlaboratory validation studies, and not trueness or whichever other method performance parameter. Evalu-... 

Validation attempts in tlie field of drug residue analysis have demonstrated that die requirement for a full collaborative trial at the ideal level, while desirable, is sometimes impractical. Limiting factors for completing ideal multilaboratory validation studies are usually the high cost, lack of sufficient expert laboratories willing to participate in such studies, and overall time constraints. Hence, a three-laboratory validation study is often applied (16). 

Provision of a unified and disciplined framework that covers all aspects of the validation process from sample and method selection to full collaborative trial. 

The overall process of method validation is illustrated in Figure I. However, the extent and scope of validation is governed by the applicability of the method. An in-house procedure requires a less exacting process than a method intended for multi-matrix and/or multi-laboratory use. For the latter methods, a full collaborative trial is necessary and is covered in Chapter 9. However, for many purposes validation is limited to either demonstrating that method performance criteria established during development are met under routine laboratory conditions and/or showing method equivalence (Figure 18). 

Why is it that many published methods of analysis do not work when they are applied outside the developer s laboratory Why do so many collaborative trials fail to produce consistent results Some of the reasons may lie with inadequacy of method validation, sample preparation or lack of specification of key analytical parameters. However, even for well developed procedures, the failure... 

As mentioned in the previous section, analytical methods intended for interlaboratory use should be subjected to a collaborative trial using the lUPAC protocol. This is a prescriptive procedure and outlier testing/removal is mandatory. Before the process begins the data need to be examined to ensure that only valid data are input to the calculation process. This process is best shown as a flow chart (Figure 36). 

Following the success of these collaborative trials, standard methodologies for the application of the EPR method for the identification of irradiated meat bones, fish bones and some fruits have been prepared and are about to be submitted to the European Committee of Normalization. At the time when these trials were carried out there was not sufficient information available to permit inclusion of Crustacea among the products being examined and thus validation of the method for the identification of these irradiated foods has to be undertaken. 

It is important that there is a low level of doubt about the identity of the compound being measured and a high level of certainty that the quantity determined is a true reflection of the amount actually present. To achieve this, confirmatory techniques usually employ complex separation procedures to isolate the compound of interest and require calibration procedures that involve adding known amounts of the compound to uncontaminated specimens of the material under analysis. As a final check on the validity of the method it is recommended that the method should be evaluated in a collaborative trial in a minimum of three independent laboratories against a standard reference material. 

The method developed in the BCR project (Franz and Rijk 1997) to determine butadiene in all of the official food simulants and probably also in real foodstuffs was pre-validated by a collaborative trial with three laboratories. It was found appropriate in principle for the quantitative determination of butadiene at a range of 0.01 to 0.1 mg/kg in food simulants. Indeed the limit of detection was found to be in the range 4 to 9 (J-g/kg, thus being even in the worst case significantly lower than originally presumed when establishing the Plastics Directive limit of 0.02 mg/kg. 

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]. 

The repeatability and within-laboratory reproducibility (expressed as CV percent) are evaluated against the Horwitz equation according to the EU Commission Decision [4]. The Horwitz equation is an empirical relationship between the concentration of the analyte and the precision of the method. Initially the Horwitz equation was developed from data obtained during collaborative trials [38, 39]. The following equation for the maximum reproducibility CV is valid. The maximum repeatability is between one-half and two-thirds of the CVR (percent). 

Azen 1979 Massart et al. 1990 Gardiner 1997). This random effects model is often used in analytical chemistry to breakdown a total precision into its components such as between-days and within-days, or between-laboratories and within-laboratories in collaborative trials, to validate an analytical method using reference material. 

Abstract ISO principles of measurement uncertainty estimation are compared with protocols for method development and validation by collaborative trial and concomitant top-down estimation of uncertainty. It is shown that there is substantial commonality between the two procedures. In particular, both require a careful consideration and study of the main effects on the re-... 

Due to the demand for reliable and comparable methods, performance requirements have been established at a national and international level for implementation of official methods, e.g. by European legislation, by the CEN or the Association of the Analytical Communities (AOAC) International, and worldwide by Codex Alimen-tarius (CAC). Thus any method proposed to be used for official purposes must be validated in a collaborative trial study, resulting in defined method performance characteristics [4], The framework for the design and conduct of such collaborative trial studies, as well as the statistical evaluation, are also defined in appropriate protocols [5]. Any method that has been successfully validated according to these protocols can be recognised as an official method for use in legal cases or for international trade control purposes. 

Food methods validated by a collaborative trial study and those validated using the single-laboratory approach have been adopted as national and international standards by, e.g. CEN, International Organisation for Standardisation (ISO), AOAC International and by the Joint FAO/WHO Codex Alimentarius Food Standards Programme. A number of EN Standards developed by CEN relate to the organisation of controls. It is however important to keep in mind that, in addition to the method performance criteria, economical and prevention strategy... 

The IRMM has recently started the validation of presently available methods (mostly based on enzyme-linked immunosorbent assays) in a collaborative trial study with real food samples such as cookies and dark chocolate containing peanuts in minute amounts (1-20 mg/kg). These methods have been already been validated in-house [20]. 

So, the methods characteristic of each test, comprising taken together a type of tests, must undergo validation testing of their results. This is the implementation of the method, and the establishment of a standard for its performance. For the standardization of quantitative methods, this consists at a minimum of a determination of trueness when blank utilization, certified reference materials (or reference materials, or spiking materials) or collaborative trials are used, repeatability (r) with repetition,... 

The aim of this trial was to validate MARA by evaluating the performance of the assay at different laboratories worldwide. The method of implementation of the testing could have been considered in the same perspective as a collaborative trial used to validate a standard method. The trial was viewed with potential to form the basis of laboratory proficiency testing defined by ISO as determination of laboratory testing performance by means of inter-laboratory comparisons (Horowitz, 1988). 

STX is now distributed worldwide by IAEA and is being used in collaborative trials of the PSTs receptor binding assay (RBA). Single laboratory validation of the RBA using new radiolabeled saxitoxins were presented at the Marine and Freshwater Toxins Analysis First Joint Symposium and AO AC Task Force Meeting in Baiona, Spain, in April 2005. The limit of quantitation of the microplate format assay " was found to be 1.2 pg STX equivalent/100 g shellfish (regulatory limit 80 pg/100 g), with an overall repeatability of 17.7% for shellfish extracts run by one analyst on 5 independent days, and a correlation r =. 98 with the mouse bioassay. 

This document was updated and replaced in 2009 by CAC/GL 71-2009. This new document introduced a number of major revisions to previous practice, particularly the introduction of performance criteria for analytical methods and the principle of single-laboratory validation. In the earlier document, analytical methods were accepted by the CCRVDF only if they had been fully validated by a rigorous multi-laboratory collaborative trial. Experience within the CCRVDF had shown that it was becoming increasingly difficult to organise such trials, and many laboratories were adopting performance criteria to validate analytical methods. 

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