Method validation testing

Table 4.42. Raw Data from Method Validation Tests...

There are several reasons for careful placement of the ruggedness test in a program of method validation tests. Firstly the ruggedness test itself can be a complex and time consuming task and thus should be carried out as late in the method validation as possible, (i.e. when most other performance characteristics have been established and are acceptable). This reduces the chance of a failed ruggedness test and for this reason it is recommended that the precision study be one of the last experiments in a validation study. 

Select a total QC strategy to provide an appropriate balance between statistical and nonstatistical QC procedures. With 90% error detection, depend on the statistical QC component and perform the minimal preventive maintenance, instrument function checks, and method validation tests required by good laboratory practice, manufacturers instructions, and regulatory and accreditation guidelines. 

Key words In vitro method, Test method, Validation, Test system, Reference item, Control item,... 

With some products, particularly snap beans, there may be some reason to question the validity of a negative catalase reaction as a criterion of adequate blanching, especially in the light of the work reported by Bedford and Joslyn (3). In the case of peas, however, it seems to be entirely adequate. There is urgent need for investigation and the development of an adequate method for testing adequacy of blanching in finished products. 

We have said that every time the calibration analyzes a new unknown sample, this amounts to an additional validation test of the calibration. It can be a major mistake to believe that, just because a calibration worked well when it was being developed, it will continue to produce reliable results from that point on. When we discussed the requirements for a training set, we said that collection of samples in the training set must, as a group, be representative in all ways of the unknowns that will be analyzed by the calibration. If this condition is not met, then the calibration is invalid and cannot be expected to produce reliable results. Any change in the process, the instrument, or the measurement procedure which introduces changes into the data measured on an unknown will violate this condition and invalidate the method If this occurs, the concentration values that the calibration predicts for unknown samples are completely unreliable We must therefore have a plan and procedures in place that will insure that we are alerted if such a condition should arise. 

Control All control points starting with the basic raw materials right through to the finished product must be identified. Descriptions of the specifications, test methods, reference standards, and methods validation data should be included. 

The method of test functions is quite applicable in verifying convergence and determining the order of accuracy and is stipulated by a proper choice of the function I7(x). Such a function is free to be chosen in any convenient way so as to provide the validity of the continuity conditions at every discontinuity point of coefficients. By inserting it in equation (1) of Section 1 we are led to the right-hand side / = kU ) — qU and the boundary values jj, — U(0) and = U 1). The solution of such a problem relies on scheme (4) of Section 1 and then the difference solution will be compared with a known function U x) on various grids. 

Established in 1894, AOAC International is an independent association of scientists and organizations in the public and private sectors devoted to promoting methods validation and quality measurements in the analytical sciences. AOAC has a mission to ensure the development, testing, validation, and publication of reliable chemical and biological methods of analysis for foods, drugs, feed-stuffs, fertilizers, pesticides, water, forensic materials and other substances affecting public health and safety and the environment. 

In contrast to the requirements for enforcement methods, validation of a previously collaboratively tested method, which is used to generate data, should be validated for new laboratory conditions. Also, where published methods are submitted, validation is required, when applied to the relevant sample matrix and laboratory conditions. 

For multi-analyte and/or multi-matrix methods, it is not possible to validate a method for all combinations of analyte, concentration and type of sample matrix that may be encountered in subsequent use of the method. On the other hand, the standards EN1528 andEN 12393 consist of a range of old multi-residue methods. The working principles of these methods are accepted not only in Europe, but all over the world. Most often these methods are based on extractions with acetone, acetonitrile, ethyl acetate or n-hexane. Subsequent cleanup steps are based on solvent partition steps and size exclusion or adsorption chromatography on Florisil, silica gel or alumina. Each solvent and each cleanup step has been successfully applied to hundreds of pesticides and tested in countless method validation studies. The selectivity and sensitivity of GC combined with electron capture, nitrogen-phosphorus, flame photometric or mass spectrometric detectors for a large number of pesticides are acceptable. 

Many experts in Europe have tested the methods of both standards with various pesticide-matrix combinations in their own laboratories. Consequently, the responsible working groups of CEN TC 275 concluded that these are the best methods available. Nevertheless, there is no complete validation of all possible pesticide-matrix combinations. However, for most multi-residue methods within the standards all those pesticides which had been successfully tested in method validation trials and/or proficiency tests are listed. Also, matrices which had been examined in ring tests are listed. 

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 analyte stability during storage and processing of samples or in standard solutions and extracts is not part of method validation in Germany. Therefore, insufficient stability will not be routinely detected and even then more or less only by chance. Also, separate tests for analyte homogeneity and extraction efficiency were not performed. 

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. 

Table 14 can be regarded as providing a reasonable overall picture, even if the results cannot applied to any particular case. However, if the underlying principle is accepted, it becomes clear that improvements in a single stage, for example the reduction of instrument variation, has a negligible beneficial effect (if this variation was not outside the normal range ). Even if the contribution of repeatability is re-duced to zero, the cumulative uncertainty is reduced by 10% only, i.e. from 2.2 to y(0.0)2 (0.8)2 (1.0)2 + (1.5)2 = 2.0. This statistical view of errors should help to avoid some unnecessary efforts to improve, e.g., calibration. Additionally, this broad view on all sources of error may help to detect the most important ones. Consequently, without participation in proficiency tests, any method validation will remain incomplete. 

Untreated (control) soil is collected to determine the presence of substances that may interfere with the measurement of target analytes. Control soil is also necessary for analytical recovery determinations made using laboratory-fortified samples. Thus, basic field study design divides the test area into one or more treated plots and an untreated control plot. Unlike the treated plots, the untreated control is typically not replicated but must be sufficiently large to provide soil for characterization, analytical method validation, and quality control. To prevent spray drift on to the control area and other potential forms of contamination, the control area is positioned > 15 m away and upwind of the treated plot, relative to prevailing wind patterns. 

Method validation is needed to demonstrate the acceptability of the analytical method. A recovery test on a chemical being determined should be performed in order to verify the reliability of the series of analyses. Recovery studies are usually conducted by spiking untreated sediment with the target chemical at the deteetion limit, quantitation limit and in the range of 10-50 times the detection limit. The method is considered acceptable when the recoveries typically are greater than 70%. When the recovery is less than 70%, an improvement in the analytical methods is needed. Where this is not possible for technical reasons, then lower recovery levels may be acceptable provided that method validation has demonstrated that reproducible recoveries are obtained at a lower level of recovery. Analysis is usually done in duplicate or more, and the coefficient of variation (CV) should be less than 10% to ensure that recoveries will be consistently within the range 70-110%. 

Once you have confidence that your method is adequate from the preliminary work in the method tryout, you are ready to begin the method validation. The method validation provides additional data on accuracy and precision, and confirms that there are no problems due to interference. Method validation must be completed before beginning the analysis of the treated samples from the field. The validation should test the detector s response over the expected range of concentrations from the field. 

Shifflet, M. J. and Shapiro, M., Validation of a cation-exchange method to test for residual amounts of a cleaning and sanitizing solution on pharmaceutical process equipment, BioPharm, 51, Jan. 2000. 

Endpoint Non-animal testing method Validation/regulation... 

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