Method parameters

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters this provides an indication of its reliability during normal usage. 

If this criterion cannot be met, optimize the instmment operating parameters or change the instrument method parameters such as injection size until a signal-to-noise ratio of 9 1 is obtained. 

The only published immunoassay method submitted to date to EPA OPPTS as an enforcement method for a range of substrates (water, sediment, crops, processed crop fractions, and animal tissues) is the spinosad method, developed by Young et al. This method uses the spinosad RaPID Assay (Strategic Diagnostics) for determination of total spinosad residues (TSR). This discussion will be limited to crop and animal tissues, because the water and soil analyses are analogous to the triasulfuron method. The extraction, cleanup, and method parameters are summarized in Table 2. 

The robustness of an analytical method can be defined as a measure of the capability of the method to remain unaffected by small, but deliberate, variations in method parameters. The parameter therefore provides an indication of the method reliability during normal usage. The ruggedness of a method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions, such as different laboratories, different analysts, different instruments, different lot of reagents, different days, etc. 

For all MicroSYNTH systems, reactions are monitored through an external control terminal utilizing the Easy WAVE software packages. The runs can be controlled by adjusting either the temperature, the pressure, or the microwave power output in a defined program of up to ten steps. The software enables on-line modification of any method parameter and the reaction process is monitored through an appropriate graphical interface. An included solvent library and electronic lab journal feature simplifies the experimental documentation. 

Experiment number Method parameter Observed result... 

Another feature of the control software should be the ability not to download the method at the beginning of every run unless the method changes. Each time the control software downloads method parameters, typically 2 to 5 sec are consumed in a situation where every second counts. The more complex the instrument is (second pump, switching valves, additional detectors), the longer the initialization phase will be before a run starts. 

Therefore, the development and validation of a scientifically sound dissolution method requires the selection of key method parameters that provide accurate, reproducible data that are appropriate for the intended application of the methodology. It is important to note that while more extensive dissolution methodologies may be required for bioequivalency evaluations or biowaivers (i.e., multiple media, more complex dissolution media additives, and multiple sampling time points), it is also essential for the simplified, routine quality control dissolution method to discriminate batch-to-batch differences that might affect the product s in vivo performance. 

In the early stages of new product development, it may not be necessary to perform all of the various validation studies. However, the process of validating a method cannot be separated from the actual development of the method conditions, because the developer will not know whether the method conditions are acceptable until validation studies are performed. The development and validation of a new analytical method may therefore be an iterative process. Results of validation studies may indicate that a change in the procedure is necessary, which may then require revalidation. During each validation study, key method parameters are determined and then used for all subsequent validation steps. 

For early phase methods emphasis is placed on specificity and these methods generally require less extensive validation than those in final development. The following method parameters should be included in... 

A method is considered to be robust when the effect of typical fluctuations in the method parameters on the outcome of the procedure is demonstrated to be negligible. Robustness is demonstrated by a test in which the effect of deliberate changes in the method parameters is studied on a number of method responses. The extent of the changes should... 

A statistical significant effect is not always relevant from the practical point of view. Therefore, a worst-case level combination experiment with regard to the studied response (e.g., resolution) is determined and performed with replicates. In this experiment, only the method parameters with major effects (both statistically significant and almost significant) are considered. As can be seen in Figure 15, the major effect, temperature and pH, are easily detected. The worst-case combinations... 

Chapter 6 describes the desired improvement of method parameters from the robustness point of view, not from the analyte or the specific analysis point of view. Illustrative examples are provided to help the reader develop methods with the currently available equipment. It is important that the method be described explicitly and unequivocally and that the validation report does not raise expectations that cannot be met in daily use. 

This chapter discusses general considerations for improving capillary electrophoresis (CE) method performance from a robustness angle. Several method parameters are discussed and examples are offered of how CE procedures are handled to obtain optimal performance. The purpose of this chapter is to raise the awareness and help the development of more robust and sensitive methods. 

Before starting any method development, you have to know the method purpose, and from the method purpose you have to define the performance demands. To improve the performance of CE methods, parameters such as instrumental settings, the injection procedure, the composition and preparation of the BGE, sample, and standards all need to be considered carefully. It is important that the final method is described explicitly and unequivocally in all aspects. 

To our knowledge, in the first approach described, Youden and Steiner introduced the term ruggedness test for a setup in which by means of an experimental design the influences of minor but deliberate and controlled changes in the method parameters or factors are evaluated in order to detect non-robust factors, i.e., with a large influence on the response. Controlling the latter factors avoids problems in the following interlaboratory study. 

The ICH guidelines define robustness as The robusmess of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.Thus, the robustness of a method is a measure of its capacity to be transferred to another laboratory without affecting the analytical result. A robustness test is the experimental setup applied to evaluate the robustness of the method. The ICH guidelines also state that One consequence... 

During the method development, key method parameters are determined and used for subsequent validation steps to ensure that the validation data are generated under conditions equivalent to the final procedure (risk analysis).Aims of the method development are summarized in the list that follows. 

A validation protocol adapted from the experiences during the method development defines the scope of the validation study (goal of the study, regulating guidelines, key method parameters, etc.). To investigate the adequate method performance, these features (e.g., range of analyte concentration), together with a statement of any fitness-for-purpose criteria, have to be specified in the validation protocol. A basic check has to provide that the reasonable assumptions about the principles of the method are not seriously flawed. In this process, sources of error in analysis have to be listed (Table 4) and their effects have to be checked. The validation should, as far as possible, be conducted to provide a realistic survey of the number... 

Robustness Capacity to remain unaffected by small, but deliberate, variations in method parameters ... 

Before robustness testing is started, precise specification of all method parameters is certainly needed (Table 6) else it may not be clear later which method has been validated. Possible settings for initial CE experiments are shown in Diagram 2. In particular, it is important to specify the buffer composition, e.g., by buffer recipes (Table 8). Otherwise, inadvertent variations in pH or ionic strength can lead to variability in selectivity. 

The evaluation of robustness should be considered in the development of the assay and will depend on the type of procedure under development. It must show the reliability of a method with respect to deliberate variations in method parameters. If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement might be included in the procedure. One consequence of the evaluation of robustness may be that a series of system suitability parameters is established to ensure that the validity of the analytical procedure is maintained whenever used. Typical parameters to be tested would be the following sample concentration, sample stability, labeling variability (if applicable), injection variability, reagent lot-to-lot variability, and capillary vendor. 

In summary, the following method parameters must be considered when selecting factors and their levels for a HPLC ruggedness test [14] ... 

The evaluation of robustness is normally considered during the development phase and depends on the type of procedure under study. Experimental design (e.g., fractional factorial design or Plackett-Burman design) is common and useful to investigate multiple parameters simultaneously. The result will help to identify critical parameters that will affect the performance of the method. Common method parameters that can affect the analytical procedure should be considered based on the analytical technique and properties of the samples ... 

The traditional criteria approach is to identify specific performance parameters and to assign numeric values to these. These numeric values represent cutoff or threshold values the method parameters must meet in order for the method to be acceptable. The alternative approach is focused on fitness for purpose and MU. In this fitness-for-purpose approach, the overall MU is estimated as a function of the analyte concentration (see Section 8.2.2). 

Ability of method to remain unaffected by small variations in method parameters (some authors make the distinction be-between the property robustness and a ruggedness test in which deliberate changes are made in a method to assess the robustness)... 

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... 

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