Inter-laboratory method validation

Within CEN TC 275, a distinct protocol for the conduct of inter-laboratory method validation trials is not selected or required, but it is recommended to apply ISO 5725 or the ISO/International Union for Pure and Applied Chemistry (lUPAC) harmonized protocol. " In the past, results of several national and international interlaboratory trials had been accepted. Nevertheless, the following fundamental aspects of the harmonized protocol for the design of method-performance studies should be fulfilled ... 

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. 

The integration of analytical methods in European standards requires their acceptance by several national experts within special working groups and in a final weighted vote of National Standards Bodies. Therefore, there needs to be very high confidence in the performance of methods. Consequently, methods should be tested in inter-laboratory method validation studies, with the exception of those multiresidue methods which are widely used throughout Europe. In the case of CEN methods there is no doubt about residue definition but detailed requirements about the number of matrices and concentration levels in validation experiments do not exist. Eor this reason it may be that CEN methods are validated for important crops only. 

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). 

During method development (Chapter 9) and validation (Chapter 10), QCs are used for several purposes including checks on precision and accuracy, lower limit of quantitation (LLOQ), recovery and method robustness and ruggedness (Section 9.8.4), as well as stability studies of various kinds (Sections 10.2.7 and 10.2.8), studies of inter-day validation within a specified laboratory and cross-validations in inter-laboratory method transfer (Section 10.2.11). QC samples are also used during method development to assess the final method prior to validation experimental runs that use QCs for this purpose are often referred to as assay prequalifications or pre-study assay evaluations (PSAE). 

Important part of validation procedure is prognosis (on basis of Phai macopoeial requirements and results of inter-laboratory trials) of sample preparation, final analytical operation and total uncertainties. It enables to forecast method uncertainty in control laboratories. 

The use of reference samples for method calibration and development/validation occurred hand-in-hand with the development of all modern instrumental methods of analysis. In fact, the two developments are intimately linked with one another. As already noted, G-i and W-i (Fairbaim et al. 1951 Stevens i960) illustrate first instance of reference samples specifically developed for calibration purposes. Following that, the use of BCR-i as a reference sample throughout the lunar program (Science 1970) is a prime illustration of the quality assurance and method validation applications in large-scale inter-laboratory measurement programs. 

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). 

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. 

In summary, the procedure of the Nordic Committee describes a comprehensive validation protocol, but it is not specially designed for pesticide residue analysis and has no preferences with regard to single- or inter-laboratory validation. Therefore, if it is applied to pesticide residue methods, some specific validation requirements should be added. The procedure clearly lists all necessary steps of validation and adjusts its recommendations to the degree of previous external validation. 

In summary, official German analytical methods for pesticide residues are always validated in several laboratories. These inter-laboratory studies avoid the acceptance of methods which cannot readily be reproduced in further laboratories and they do improve the ruggedness of analytical procedures applied. The recently introduced calibration with standards in matrix improves the trueness of the reported recovery data. Other aspects of validation (sample processing, analyte stability, extraction efficiency) are not considered. 

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. 

In Europe, very different concepts of method validation are in use. The extent of validation depends upon legal requirements (e.g., for enforcement methods provided by the applicant), upon the required level of acceptance (e.g., for CEN methods) and upon national resources. Undoubtedly, the best method validation is performed with the help of inter-laboratory studies of performance, but such studies can be uneconomic, too slow to reach completion or restricted in scope. 

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... 

However, other approaches may be equally valid. For instance, some inter-laboratory assessments utilise samples that have been spiked with a known concentration of an impurity(ies). Thus if the receiving laboratory meets the pre-determined acceptance criterion this constitutes an acceptable transfer. The transfer protocol is pre-approved by both transferring and receiving laboratory(ies) and stipulates all of the details of methodology, samples, acceptance criterion and appropriate method variability. 

Laboratories that participate in the validation of a method are qualified to perform the procedure (USP, 2012c). Thus, the transferring laboratory can involve the receiving laboratory in an inter-laboratory(ies) co-validation exercise of the reproducibility of the method. Typically, the key parameters to be assessed are RDSip, the assessment of specificity within the receiving laboratory and for impurity methods, the assessment of LOD/LOQ are recommended (Agut et al., 2011). As with comparative testing, a pre-approved... 

To facilitate a standardisation of inter-laboratory results of permeability, it is now common practice to include a range of model drugs as internal standards in initial validation (i.e. method suitability) of intestinal perfusion techniques [116]. A list of 20 model drugs has been reported by the FDA for the standardisation of the in situ intestinal perfusion experiment, whereas six drugs are recommended for human studies. Once the method has been... 

Methods have successfully been transferred to various laboratories in inter-company cross-validation exercises for a chiral separation, for an assay of the main component in a formulation and for drug stoichiometry. Revalidation is an alternative to method transfer in case of changes in product composition or analytical procedure (cf. Section I.L). Although a method transfer in CE is not a major difficulty, some aspects have to be considered, especially if a method is transferred to an instrument of another manufacturer. 

At the same time. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) evaluated FETAX methodology based on the inter-laboratory studies and published a review document in 2000 (5). The expert panel concluded that FETAX was not sufficiently validated for regulatory use due to the intra- and inter-laboratory variability (6). Nonetheless, the assay was developed in our laboratory for use as a predictive screening assay. 

In analytical chemistry, validation of the analytical methods is of utmost importance [4,5]. One of the aspects of this validation is the robustness of analytical methods against variations in experimental circumstances. The term experimental circumstances is very broad it might even include inter-laboratory variation. In this book, only intra-laboratory experimental conditions are considered. No explicit attention is given to inter-laboratory variations, although some of the presented methodology might be useful in that area. 

Beyond test development and validation, inter-calibration exercises (also known as round robin or inter-laboratory exercises ) are mandatory steps that must be undertaken if a toxicity test method is intended for standardization. These exercises... 

In their regular day to day practice, field laboratories use commercial reagents or prepare in-house solutions for the calibration of instruments, and they rely on purity assessment of producers. For method validation and even measurement uncertainty, field labs regularly participate in proficiency testing schemes. In such inter-laboratory comparisons, the reference value is usually obtained as the arithmetic mean of results of participants. 

Once a method is established, precision may be determined by suitable replicated experiments. However it is in inter-laboratory trails that the problems with environmental methods often show up. It is accepted that for trace analysis RSD values of tens of percent are likely. In studies conducted in Western Australia on pesticide residues in bovine fat RSD values for dieldrin were 12% and for dia-zonium were 28%. It is typical to see a quarter of the laboratories in such a trial producing values that could be termed outliers. In the previously mentioned study, 5 laboratories out of 26 had z> 3 for aldrin. In a parallel study RSD values for petroleum in dichloromethane and water were 40% and 25%, respectively. The conclusions of these studies was that there was poor comparability because of the different methods used, that accreditation apparently made no difference to the quality of results, and that a lack of understanding of definitions of the quantities to be analysed (for example gasoline range organics ) caused non-method errors. In relation to methods, this is contrary to the conclusion of van Nevel et al. who asserted that the results of the IMEP round of analyses of trace elements in natural and synthetic waters showed no dependence on method [11]. If properly validated methods do yield comparable results, then one conclusion from the range of studies around the world is that many environmental methods are not validated. It may be that validated methods are indeed used, but not for exactly the systems for which they were validated. 

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