Validation experiments

This can really be seen as being an assessment of analytical methods in the presence of four different types of components. It is particularly important, and most often challenging, in ligand-binding methods, which do, of course, form the majority of biomarker assays used in drug development today. 

All of these interferences can be evaluated by spiking in the compounds of interest at concentrations that are likely to be encountered. The authors commonly test for up to two or three times these concentrations to allow some safety margin for possible specimens where they may be present at higher levels than expected. 

It should be recognized that full specificity of LB A methods for macromolecules (e.g., proteins, oligonucleotides, etc.) can never be demonstrated for most assays because of a lack of knowledge of the structures and cross-reactivities/interferences of intermediate products of metabolism or the availability of these to test. Therefore, it is important for scientists working in this arena to appreciate the full range of challenges related to method specificity some of which may not have been previously encountered within their analytical field of work. 

Commercial kits are usually supplied with calibration standard material. This may be in the form of a single vial of product from which a calibration curve can be constructed by serial dilution, or it may be a series of vials of ready-to-use standards with concentrations representative of the method s analytical range. For the purposes of assays used in drug development, it is often the case that the user will require a larger amount of calibration material, or a larger number of calibrator levels than is supplied. In the latter case, it is often possible to produce additional calibration standards from those provided either by dilution or by the mixing of those supplied. 

It should be understood that in most cases, the calibration material provided may not be 100% pure (most are not supplied with a Certificate of Analysis). However, notwithstanding the recommendations below, this is often not a major issue since the majority of biomarker assays will, by definition, be relative quantitative assays that do not measure absolute concentration. In addition, in many assay kits, particularly more recent ones, the calibration material may not be well characterized or purified, or it may not be fully representative of the endogenous analyte to be measured. Therefore, we would recommend procuring material from at least one other third-party manufacturer against which concentrations can be checked to ensure consensus. If there is disparity, another source may be required. It is also a good idea to check with the kit manufacturer as to where they have sourced their calibration material, since they often do not manufacture it themselves. On more than one occasion with certain assays we 

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The following flowsheet represents the simplest connections combined with good, inexpensive manual regulation required to execute valid experiments. This is the recommended minimum starting installation that can be expanded and made more sophisticated as need and budgets permit. The other extreme, a fully computer controlled and evaluated system that can be run without personnel will be shown later. The concepts, mentioned in Chapter 3, are applied here for the practical execution of experiments in recycle reactors. 

Figure 4.31. Key statistical indicators for validation experiments. The individual data files are marked in the first panels with the numbers 1, 2, and 3, and are in the same sequence for all groups. The lin/lin respectively log/log evaluation formats are indicated by the letters a and b. Limits of detection/quantitation cannot be calculated for the log/log format. The slopes, in percent of the average, are very similar for all three laboratories. The precision of the slopes is given as 100 t CW b)/b in [%]. The residual standard deviation follows a similar pattern as does the precision of the slope b. The LOD conforms nicely with the evaluation as required by the FDA. The calibration-design sensitive LOQ puts an upper bound on the estimates. The XI5% analysis can be high, particularly if the intercept should be negative.

The validity of the model is tested against the experiment. A ISOOcc canister, which is produced by UNICK Ltd. in Korea, is used for model validation experiment. In the case of adsorption, 2.4//min butane and 2.4//min N2 as a carrier gas simultaneously enter the canister and 2.1//min air flows into canister with a reverse direction during desorption. These are the same conditions as the products feasibility test of UNICK Ltd. The comparison between the simulation and experiment showed the validity of our model as in Fig. 5. The amount of fuel gas in the canister can be predicted with reasonable accuracy. Thus, the developed model is shown to be effective to simulate the behavior of adsorption/desorption of actual ORVR system. 

Enforcement methods provided by the manufacturer are not generally tested in the laboratories of the European regulatory authorities. Very often, proposed methods are evaluated by assessing the logic of proposed procedures and only for the completeness of validation data. For this theoretical review process, as much information as possible should be available. Recovery data from many validation experiments with different kinds of matrices and the resulting chromatograms of control and fortified samples provide the confidence needed by the referee. In the following sections, the most important aspects of this evaluation will be considered. 

Table 2 Matrix-study combinations for which method validation experiments are needed...

The rationale of validation experiments with fatty matrices is the high amount of fat extracted with many organic solvents. If analytes are not fat soluble and extraction is performed with water or aqueous buffer solutions, the troublesome fat is not extracted together with the analyte. Such extractions are typical for, e.g., the class of sulfonylurea herbicides. Examples exist where in such cases the applicability of an analytical method to fatty matrices was accepted by the authority without particular validation. 

Crops with high acid content have to be tested separately, to demonstrate the robustness of methods with regard to changes in pH. In such cases, where extractions are performed at pH values which are lower than those of acidic crops (e.g., <3), the influence of sample acidity is not significant. It is assumed that under such circumstances an expert statement should be sufficient and may replace validation experiments with representative commodities of this matrix group. 

A final special case may occur during the validation of common moiety methods. Based on the normal set of recovery experiments (two control samples, five samples fortified at the LOQ and five samples fortified at 10 times the LOQ), in total 12 samples have to be analyzed per matrix and analyte. A typical intention of common moiety methods is their suitability for the parallel determination of residues of the parent compound and a broad spectrum of metabolites. In the common moiety method discussed above for residues of spiroxamine, validation experiments were performed with four compounds. This results in at least 48 experiments per matrix. Assuming a normal... 

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. 

To validate the skin carotenoid RRS detection approach, we initially carried out an indirect validation experiment that compared HPLC derived carotenoid levels of fasting serum with RRS derived carotenoid levels for inner palm tissue sites. Measuring a large group of 104 healthy male and female human volunteers, we obtained a significant correlation (p < 0.001) with a correlation coefficient of 0.78 (Smidt et al. 2004). Recently, we carried out a direct validation study, in which we compared in vivo RRS carotenoid skin responses with HPLC-derived results, using the thick... 

It appears as if an axiom of stereochemistry, the absolute identity of the most important chemical and physical properties of chiral isomers, is no longer valid. Experiments using the amino acid tyrosine (Tyr) showed unexpected differences in the solubility of D-and L-Tyr in water a supersaturated solution of 10 mM L-Tyr crystallised much more slowly than that of D-Tyr under the same conditions. The saturated solution of L-Tyr was more concentrated than that of D-Tyr. Supersaturated solutions of DL-Tyr in water formed precipitates containing mainly D-Tyr and DL-Tyr, so that there was an excess of L-Tyr in the saturated solution. The experiments were carried out with extremely great care in order to exclude the possibility of contamination. Further experiments will show whether this is a particular property of tyrosine, or whether other amino acids will show similar behaviour. Possible... 

As a validation experiment a large number of load speed measurements on Twaron 2200 PpPTA yarn at different temperatures have been carried out. In order to limit the scatter of the data a slight twist was applied to the yarn. Figure 67 shows the fit of the linear relation Eq. 135 with the experimental data. The values for the parameters used in this fit are listed in Table 5. As stated earlier, the linear relationship Eq. 135 was derived for the approximation t<2g. According to Eq. 134, for large values of the load rate the second term should become very small. Indeed, in Fig. 67 the observed data tend to level off for... 

This method, specific for the epoxidation of allylic alcohols, gives good results if the reaction is carried out under strictly anhydrous conditions, otherwise the yield or the enantiomeric excess can decrease, sometimes dramatically. This can explain the small differences between the results obtained during the validation experiments and the published results. All the different reagents are commercially available they can be used as received but in case of low yield and/or enantiomeric excess they should be distilled and dried under an inert atmosphere. Table 5.1 gives some other examples of substrates which can be epoxidized using the procedure described above. 

The process of method development and validation covers all aspects of the analytical procedure and the best way to minimize method problems is to perform validation experiments during development. To perform validation studies, the approach should be viewed with the understanding that validation requirements are continually changing and vary widely, depending on the type of product under test and compliance with any necessary regulatory group. 

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. 

System suitability test characteristics and limits are recommended as a component of any analytical method. This ensures that both methodology and instrumentation are performing within expectations prior to the analysis of test samples. The test characteristics are inferred from robustness studies and evaluated during the validation experiments. 

A number of computer software packages are available to the analyst to assist in the planning and execution of both method development and validation experiments. The attraction of these systems is that they can automate the validation process from planning the experiment to test execution to the presentation of the data in a final report form. 

Comparisons as in Fig. 6.31 serve as tool to improve and vahdate MD simulation results and methods and will help to develop more efficient simulation methods. The interplay between validating experiments and successively improved simulations is a very promising approach for arriving at a very detailed picture of the internal motion within biopolymers. 

The factors that influence the optimal cross-validation method, as well as the parameters for that method, are the number of calibration samples (AO, the arrangement order of the samples in the calibration data set, whether the samples arise from a design of experiments (DOE, Section 12.2.6), the presence or absence of replicate samples, and the specific objective of the cross-validation experiment. In addition, there are two traps that one needs to be aware of when setting up a cross-validation experiment. 

COMPOSITIONS OF THE EXTRACTION LIQUIDS USED FOR THE ALGORITHM VALIDATION EXPERIMENT AND FOR THE MODELLING OF THE EXTRACTION OF A NUMBER OF SULPHONAMIDES... 

A trial that demonstrates superiority of the new drug over placebo, plus superiority of the standard drug over placebo, has demonstrated its assay sensitivity and may be considered a valid experiment. 

The method validation experiments should be well planned and laid out to ensure efficient use of time and resources during execution of the method validation. The best way to ensure a well-planned validation study is to write a method validation protocol that will be reviewed and signed by the appropriate person (e.g., laboratory management and quality assurance). 

System suitability is part of method validation. Experience gained during method development will give insights to help determine the system suitability requirements of the final method. An example is the hydrolysis of acetylsalicylic acid to salicylic acid in acidic media. Separation of this degradation peak from the analyte could be one criterion for the system suitability of an acetylsalicylic acid assay. 

Tables 2.2 and 2.3 are examples of repeatability data. Table 2.2 shows good repeatability data. However, note that the data show a slight bias below 100% (all data between 97.5 and 99.1%). This may not be an issue, as the true value of the samples and the variation of the assay may be between 97.5 and 99.1%. Table 2.3 shows two sets of data for a formulation at two dose strengths that were performed using sets of six determinations at 100% test concentration. The data indicate a definite bias and high variability for the low-strength dose formulation. It may call into question the appropriateness of the low-dose samples for the validation experiment.

Acceptance Criteria for Validation Parameter. It is highly recommended to set acceptance criteria prior to starting validation experiments. This will provide guidance to the validating scientist on the range of acceptability of the validation results. 

Method Procedure. Since the method procedure is undergoing constant modifications during method development, it is very important to define the procedure before method validation. This will ensure that the same method procedure will be used in all method validation experiments. 

System Suitability Tests. The appropriate system suitability tests should be defined before method validation (e.g., precision, resolution of critical related substances, tailing, detector sensitivity). These system suitability tests should be performed in each method validation experiments. System suitability results from the method validation experiment can be used to determine the appropriate system suitability acceptance criteria. 

System suitability. During the robustness testing of method validation, critical method parameters such as mobile phase composition and column temperature are varied to mimic the day-to-day variability. Therefore, the system suitability results from these robustness experiments should reflect the expected range for the system suitability results. As a result, system suitability results in these method validation experiments are very useful in determining the system suitability... 

Different approaches may be used to validate the sample preparation component of the dissolution test. However, it is important to understand that the objective of validation is to demonstrate that the procedure is suitable for its intended purpose. For example, one of the strategies will demonstrate the validity of different aspects of sample preparation during method development (prior to the formal method validation exercise). As a result, the final validation experiments will confirm the work done during method development. The strategy that will be followed for the method development and validation process will depend on the culture, expertise, and strategy of the analytical laboratory. 

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