Validation Inter-laboratory study

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). 

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. 

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. 

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. 

Nilsson, T. Ferrari, R. Facchetti, S. Inter-laboratory studies for the validation of solid-phase microextraction for the quantitative analysis of volatile organic compounds in aqueous samples. Analytica Chimica Acta 1997, 356 (2-3), 113-123. 

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 ... 

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). 

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. 

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. 

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. 

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... 

Solid-phase microextraction (SPME) consists of dipping a fiber into an aqueous sample to adsorb the analytes followed by thermal desorption into the carrier stream for GC, or, if the analytes are thermally labile, they can be desorbed into the mobile phase for LC. Examples of commercially available fibers include 100-qm PDMS, 65-qm Carbowax-divinylbenzene (CW-DVB), 75-qm Carboxen-polydimethylsiloxane (CX-PDMS), and 85-qm polyacrylate, the last being more suitable for the determination of triazines. The LCDs can be as low as 0.1 qgL Since the quantity of analyte adsorbed on the fiber is based on equilibrium rather than extraction, procedural recovery cannot be assessed on the basis of percentage extraction. The robustness and sensitivity of the technique were demonstrated in an inter-laboratory validation study for several parent triazines and DEA and DIA. A 65-qm CW-DVB fiber was employed for analyte adsorption followed by desorption into the injection port (split/splitless) of a gas chromatograph. The sample was adjusted to neutral pH, and sodium chloride was added to obtain a concentration of 0.3 g During continuous... 

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... 

Brady, J.F., D.P. Tierney, J.E. McFarland, and M.W. Cheung (2001). Inter-laboratory validation study of an atrazine immunoassay. J. Amer. Water Works Assoc., 93 107-114. 

Poms, R.E., Agazzi, M., Bau, A., Brochee, M., Capelletti, C., Nordgaard, J.V., and Anklam, E. 2004. Inter-laboratory validation study of five commercially different ELISA test kits for the determination of peanut residues in cookie and dark chocolate. European Commission 2004, GE/R/FSQ/D08/05/2004. 

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. 

ESCODD (2005) Establishing the background level of base oxidation in human lymphocyte DNA results of an inter-laboratory validation study. FASEBJ., 19, 82-84. 

Very little regulatory documentation directly relates to method transfer and usually where it is mentioned, it refers to cross-validation of the method between two laboratories. It is mentioned in the FDA s Bioanalytical Methods Validation Guidance [1] page 3 under Cross-Validation, 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 to establish inter-laboratory reliability. It is also mentioned in the Appendix of the same guidance that defines reproducibility as the precision between two laboratories. ... 

These assays, which have been standardized and validated at InterceU AG s Ghni-cal Immunology Laboratory, enable reliable measurements of epitope-specific T cell responses induced by vaccination. All assays were performed in compliance with Good Laboratory Practice (GLP)/Good Clinical Practice (GCP) requirements. Standardization of the blood cell isolation procedure at the different investigational sites led to a high rate of evaluable assays. However, due to the lack of inter-laboratory standardization of T cell assays, comparison of the results of this study with published data from similar trials is difficult. Cryopre-served blood cells were used, which may have resulted in a possible underestimation of T cell responses compared with assays that utilize fresh blood. 

Telliard, W.A., McCarty, H.B. and Riddick, L.S., Results of the inter-laboratory validation study of USEPA method 1613 for the analysis of tetra- through to octachlorinated dioxins and furans by isotope dilution GC-MS, Chemosphere, 27, 41-46 (1993). 

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