Protocol, method validation precision requirements

Before any method validation is started, the scope of validation must be fixed, comprising both the analytical system and the analytical requirement. A description of the analytical system includes the purpose and type of method, the type and concentration range of analyte(s) being measured, the types of material or matrices for which the method is applied, and a method protocol. On the basis of a good analysis lies a clear specification of the analytical requirement. The latter reflects the minimum fitness-for-purpose criteria or the different performance criteria the method must meet in order to solve the particular problem. For example, a minimum precision (RSD, see below) of 5% may be required or a limit of detection (LOD) of 0.1% (w/w) [2,4,15,58]. The established criteria for performance characteristics form the basis of the final acceptability of analytical data and of the validated method [58]. 

From this point the analytical department assumes leadership and begins by selecting the analytical technique. The selection process is dependent upon several related factors, including the analyte itself and the level of measurement precision required. Once the technique has been chosen, a suitable method is identified either from modification of an existing method or the development of a new method. Upon successful demonstration of method feasibility, the technical team members, in collaboration with the regulatory professionals, draft a test procedure and a validation protocol. 

We have applied this protocol to the evaluation of the measurement uncertainty for a method for the determination of three markers (Cl solvent red 24, Cl solvent yellow 124 and quinizarin (1,4-dihydroxyanthra-quinone)) in road fuel. The method requires the extraction of the markers from the sample matrix by solid phase extraction, followed by quantification by HPLC with diode array detection. The uncertainty evaluation involved four experimental studies which were also required as part of the method validation. The studies were precision, trueness (evaluated via the analysis of spiked samples) and ruggedness tests of the extraction and HPLC stages. The experiments and uncertainty calculations are described in detail in Part 2. A summary of the uncertainty budget for the method is presented in Fig. 3. 

We have developed a protocol which describes how data generated from experimental studies commonly undertaken for method validation purposes can be used in measurement uncertainty evaluation. This paper has illustrated the application of the protocol. In the example described, the uncertainty estimate for three analytes in different oil matrices was evaluated from three experimental studies, namely precision, recovery and ruggedness. These studies were required as part of the method validation, but planning the studies with uncertainty evaluation in mind allowed an uncertainty estimate to be calculated with little extra effort. A number of areas were identified where additional experimental work may be required to refine the estimates. However the necessary data could be generated by carrying out additional analyses alongside routine test samples. Again this would minimise the amount of laboratory effort required. 

When samples contain very low levels of degradation products, recovery studies are performed by spiking actual samples with known degradation products at levels consistent with the specification. Acceptance criteria for % recovery and precision should be consistent with the requirements established in the original method validation protocol. Un-spiked samples should also be run to enable correction for any amount present in the actual sample. 

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. 

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]. 

The ideal validated method would be the one that has progressed fully through a collaborative study in accordance with international protocols for the design, conduct, and interpretation of method performance studies. A typical study of a determinative method conducted in accordance with the internationally harmonized International Organization for Standardization (ISO)/International Union for Pure and Applied Chemistry (IUPAC)/AOAC International (AOAC) protocol would require a minimum of up to five test materials including blind replicates or split-level samples to assess within-laboratory repeatability parameters, and eight participating laboratories (15). Included with the intended use should be recommended performance criteria for accuracy, precision and recovery. 

In Part 1 [1] we described a protocol for the evaluation of measurement uncertainty from validation studies such as precision, trueness and ruggedness testing. In this paper we illustrate the application of the protocol to a method developed for the determination of the dyes Cl solvent red 24 and Cl solvent yellow 124, and the chemical marker quinizarin (1,4-dihydroxyanthra-quinone) in road fuel. The analysis of road fuel samples suspected of containing rebated kerosene or rebated gas oil is required as the use of rebated fuels as road fuels or extenders to road fuels is illegal. To prevent illegal use of rebated fuels, HM Customs and Excise require them to be marked. This is achieved by adding solvent red 24, solvent yellow 124 and quinizarin to the fuel. A method for the quantitation of the markers was developed in this laboratory [2]. Over a period of time the method had been adapted to improve its performance and now required re-validation and an uncertainty estimate. This paper describes the experiments under-... 

As an analytical approach to residue analysis, immunoassay methods are not well characterized, and no validation protocols have been established. The Association of Official Analytical Chemists, whose primary purpose is validation of analytical methods, established a Task Force on Test Kits and Proprietary Methods (2), which has addressed some of the issues relating to immunoassay methods. The International Union of Pure and Applied Chemistry s Commission on Food Chemistry has established a Working Group on Immunochemical Methods, whose first project is to develop draft guidelines on criteria for evaluation, validation, and quality control for r o-immunoassay methods (10). Similar guidelines for EIAs will also be developed. These documents will assist in development and standardization of requirements for precision for both between-laboratories and within-laboratory andyses, accuracy, and ruggedness, and— for qualitative methods— false positive and false negative rates. 

Prior to its use a method has to be validated. Validation is the formal proof that the method is suitable for the intended purpose. This requires that all steps and parameters of the method have been clearly specified in a written method description, any necessary equipment was qualified, and acceptance criteria for each validation point have been agreed upon. For quantitative methods the International Conference on Harmonization (ICH) has issued specific guidelines for setting up a validation protocol and for parameters that have to be validated for different applications. These include specificity, accuracy, precision, LOD, LOQ, linearity, and range as well as robustness. The only required validation parameter for qualitative methods is specificity. 

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