Validation, method concentration levels

Reproducibility in the context of Directive 96/46/EC is defined as a validation of the repeatability of recovery, from representative matrices at representative levels, by at least one laboratory, which is independent of the laboratory which initially validated the study. This independent laboratory may be within the same company, but may not be involved in the development of the method. This concept of independent laboratory validation (ILV) substitutes the conduct of interlaboratory trials (e.g., according to ISO 5725) because the resources are not available taking into consideration the high number of a.i., matrix types and concentration levels which must be validated in the registration procedure. 

The extent of validation of confirmatory techniques is currently under consideration. Qne approach is that the extent of validation may be smaller than for the enforcement method. In principle, validation in triplicate at the relevant concentration level (LOQ or MRL) is sufficient. In the case where an MRL is set for multiple crops, a single validation in all representative crop groups is sufficient. A confirmatory method for residues in air is not required if a corresponding method was submitted for the other sample matrices. This approach is realized in Germany. ... 

Today, when a pesticide with no detectable residues is registered for use, a Tolerance or maximum residue limit (MRL) is established at the lowest concentration level at which the method was validated. However, for risk assessment purposes it would be wrong to use this number in calculating the risk posed to humans by exposure to the pesticide from the consumption of the food product. This would be assuming that the amount of the pesticide present in all food products treated with the pesticide and for which no detectable residues were found is just less than the lowest level of method validation (LLMV). The assumption is wrong, but there is no better way of performing a risk assessment calculation unless the limit of detection (LOD) and limit of quantification (LOQ) of the method were clearly defined in a uniformly acceptable manner. 

Finally, it is important to define the lowest level of method validation (LLMV). The LLMV is defined as the lowest concentration level expressed in terms of amount of analyte in the matrix, at which the method (extraction/analysis procedure) was validated or proven to be capable of reliably quantifying. 

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). 

Selectivity and sensitivity of available instruments are tested in all laboratories in the initial step of validation. The crops used for fortification experiments and the concentration levels are identical in all laboratories. Recoveries are determined with all available detection techniques, but after discussion of the results each laboratory selects individually one valid result for each analyte-matrix-level combination. Only this result is used for the calculation of the final mean recovery and standard deviation. Typical criteria for the acceptance of methods are given in Table 11. 

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. 

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). 

II the difference approach, which typically utilises 2-sided statistical tests (Hartmann et al., 1998), using either the null hypothesis (H0) or the alternative hypothesis (Hi). The evaluation of the method s bias (trueness) is determined by assessing the 95% confidence intervals (Cl) of the overall average bias compared to the 0% relative bias value (or 100% recovery). If the Cl brackets the 0% bias then the trueness that the method generates acceptable data is accepted, otherwise it is rejected. For precision measurements, if the Cl brackets the maximum RSDp at each concentration level of the validation standards then the method is acceptable. Typically, RSDn> is set at <3% (Bouabidi et al., 2010),... 

III the equivalence approach, which typically compares a statistical parameters confidence interval versus pre-defined acceptance limits (Schuirmann, 1987 Hartmann et al., 1995 Kringle et al., 2001 Hartmann et al., 1994). This approach assesses whether the true value of the parameter(s) are included in their respective acceptance limits, at each concentration level of the validation standards. The 90% 2-sided Cl of the relative bias is determined at each concentration level and compared to the 2% acceptance limits. For precision measurements, if the upper limit of the 95% Cl of the RSDn> is <3% then the method is acceptable (Bouabidi et al., 2010) or,... 

In 1971 when safety and health standards were established by the U. S. Department of Labor for several hundred chemical substances, there were analytical methods available for some of the compounds, but few were validated to ensure the accurate monitoring of the exposure of workers to these toxic substances (1). Consequently, programs were undertaken by the National Institute for Occupational Safety and Health (NIOSH) to develop and validate sampling and analytical methods. The initial intent was to provide methods that would be useful to industry in measuring the exposures of personnel to potentially toxic materials at concentration levels near the accepted standard levels. Consequently, many earlier methods were developed around the standard levels established by the Occupational Safety and Health Act with validation at, for example, levels ranging from one-half to twice the established standard level (2). Often these methods were not validated at lower concentration levels, say, one-tenth of the original level. 

The error of an analytical result is related to the (in)accuracy of an analytical method and consists of a systematic component and a random component [14]. Precision and bias studies form the basis for evaluation of the accuracy of an analytical method [18]. The accuracy of results only relates to the fitness for purpose of an analytical system assessed by method validation. Reliability of results however has to do with more than method validation alone. MU is more than just a singlefigure expression of accuracy. It covers all sources of errors which are relevant for all analyte concentration levels. MU is a key indicator of both fitness for purpose and reliability of results, binding together the ideas of fitness for purpose and quality control (QC) and thus covering the whole QA system [4,37]. 

The ISO definition of validation is confirmation by examination and provision of objective evidence that the particular requirements of a specified intended use are fulfilled [15]. Method validation is needed to confirm the fitness for purpose of a particular analytical method, that is, to demonstrate that a defined method protocol, applicable to a specified type of test material and to a defined concentration rate of the analyte —the whole is called the analytical system — is fit for a particular analytical purpose [4]. This analytical purpose reflects the achievement of analytical results with an acceptable standard of accuracy. An analytical result must always be accompanied by an uncertainty statement, which determines the interpretation of the result (Figure 6). In other words, the interpretation and use of any measurement fully depend on the uncertainty (at a stated level of confidence) associated with it [8]. Validation is thus the tool used to demonstrate that a specific analytical method actually measures what it is intended to measure and thus is suitable for its intended purpose [11,55,56]. 

Typically, linearity and accuracy determination covers a wide concentration range (e.g., 50% of the ICH reporting limit to 150% of specification). However, the concentration range for precision will be limited by the availability of sample of different related substance levels. Therefore, to ensure an appropriate method validation range with respect to precision, it is critical to use samples of low and high levels of related substance in precision experiments (e.g., fresh and stressed samples). 

Method validation was carried out in six (monkey) or seven (human) validation batches. Each batch included eight levels of standard calibrators, four levels of VS at concentrations at the LLOQ, low, mid, and high concentrations within the calibrator range, and two additional concentrations higher than the ULOQ. The >ULOQ VS were run at dilution factors of 20 and 400 to mimic the expected high concentration samples. The accuracy and precision data of the VS in human plasma are listed in Table 6.3. 

Some refractory elements cannot be determined by ET-AAS at the levels usually present in waters. That is the case with M. El Himri et al. [28] developed a fast and accurate procedure, without any prior treatment, to analyze tap and mineral waters from Spain and Morocco for this highly toxic element. ICP-MS was employed. The analytical isotope selected was 238U, with Rh as internal standard. An LoD of 2ngl 1 was obtained. The estimated repeatability was 3 percent at the concentration level of 73 ng l-1. The method was validated by comparison with a radiochemical procedure devised for natural samples and by analysis of a Certified Reference Material (CRM). Multi-element capabilities of ICP-AES have also been employed for surveys of trace elements. Al-Saleh and Al-Doush [29] reported the concentrations of dissolved Be, Cd, Cr, Cu, Fe, Mg, Mn, Hg, Ni, Se, Sr, V, and Zn in 21 samples of retail bottled waters from Riyadh, Saudi Arabia. It was found that Cd, Fe, Hg, Ni, and Zn were present at concentrations higher than the limits recommended by the EU and World Health Organization (WHO) guidelines. 

The consensus values cannot automatically be accepted as recommended to certified values because their analytical validity usually requires a re-assessment in the light of additional analytical information such as concentration level, number of different analytical methods used, percent of outliers and other criteria. In practice, certified or recommended values are always based on the following requirements data should be available from a certain number of participants and two or more different analytical methods there should be no significant differences between the groups of accepted results outliers should not exceed 20-30% of the submitted results. Depending on the extent to which the data satisfy such acceptance criteria, the consensus values are then assigned to one of the following conclusions certified or recommended concentration, information value, or not recommended. 

When the analytical method has been validated for routine use, its accuracy and precision should be controlled regularly to ensure that the method continues to work satisfactorily. For this purpose, a number of separately prepared (from different weightings than the ones used for the standard curve) quality control (QC) samples should be analyzed in each run [16], The QC samples are often duplicates at three concentrations (low, medium and high) within the range. At least four of the six QC samples should be within 20% of there respectively nominal value, and at least one at each concentration level [16, 89], Also a standard curve should be processed during each run [89],... 

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