Method validation Additive precision

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. 

Further discussion of method validation can be found in Chapter 7. However, it should be noted from Table 11 that it is frequently desirable to perform validation experiments beyond ICH requirements. While ICH addresses specificity, accuracy, precision, detection limit, quantitation limit, linearity, and range, we have found it useful to additionally examine stability of solutions, reporting threshold, robustness (as detailed above), filtration, relative response factors (RRF), system suitability tests, and where applicable method comparison tests. 

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. 

Adoption of standard methods also allows gathering of extensive knowledge and validation of the methods. The method validation can initially be comprehensive including long-term buffer shelf life, instrument-to-instrument type transfer, analyst-to-analyst repeatability and extensive robustness testing. If the standard method is applied to a new solute then validation is limited and may only need to include assessment of injection precision, linearity, sensitivity, etc., which can be obtained relatively quickly and simply. Internal standards are widely used in CE to improve injection precision and to compensate for solution viscosity differences, which may unduly affect assay results. Standard internal standards can be used for specific methods, which avoids additional work selecting an appropriate choice. 

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. 

Before the performance of method transfer activities involving protocols and acceptance criteria, it was customary for a receiving laboratory to repeat some or all of the validation experiments. This laboratory was thereby deemed to be qualified as described above. The choice of validation parameter(s) depends highly on the type of method being transferred. For example, content uniformity assays to determine consistency of product potency depend heavily on the method and system precision. As a second example, a determination of trace impurities in an API could not be reproduced between two sites if their instruments did not yield similar limits of detection and limits of quantitation. A detailed discussion on the rational choice of validation parameters that would need to be repeated by the receiving laboratory is beyond the scope of this chapter. The reader is referred to the method validation chapter by Crowther et al. for additional information on this subject. 

Your instructor will select one experiment for teams to perform validation studies. An example is a gas chromatography experiment such as Experiment 32, but for one analyte. A flow injection analysis (FIA) experiment, such as Experiment 37, would be a good choice as well, since multiple measurements can be made rapidly. The team will determine linearity, accuracy, precision, sensitivity, range, limit of detection, limit of quantitation, and robustness (repeatability) of the method. In addition, a control chart will be prepared over at least one laboratory period. The instructor will have available a reference standard to use for accuracy studies. Plan for two laboratory periods for the completed study. A report of the method will be prepared and documented. Before beginning the experiment, you should review method validation in Chapter 4. 

The availability of an accurate, precise, and specific bioanalytical technique for the quantification of active drug moieties in plasma, hlood, or other hiological fluids is an essential prerequisite for the evaluation of the relationship between dose, concentration, and effect of hiotech drugs. In analogy to small molecules, these analytical techniques have to he validated and have to meet prespecified criteria regarding accuracy, precision, selectivity, sensitivity, reproducihihty, and stahihty, for example, those recommended hy the US Food and Drug Administration [10-12]. Additional requirements for bioanalytical method validation for macromolecules have recently been published [11]. 

These experiments are designated as phases II and III of a method validation or an analyst familiarization for those using the approach recommended in the USDA/FSIS Chemistry Laboratories Guidebook (see QA section of the posted methods)7 The results provide an assessment of the recovery (trueness) and the analyst precision attained with the method under routine conditions of use. In addition, the data generated may be used to calculate statistical estimates of the reliability of the results, including estimates of MU. i ... 

Parallel method comparisons are used to establish the validity of a new method developed for five organic pharmaceutical compovmds, food colors, and color additives. The standard methods such as the Japanese Standard Food Additives and Japanese Standard of Cosmetic Ingredients method, based on volumetric and gravimetric titration, have been used to establish new methods developed for the determination of I, Cl, Br, and SO4 in food colors. The results obtained indicate good agreement in both accuracy and precision for procedures based on the oxygen flask method as compared with the standard methods. In addition to anion elemental analysis, method validation has also been carried out for metal analysis such as that of Ce(III), Th(IV), and U(VI), with the results showing acceptable limits of variation. 

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