Method validation example

The process of method transfer must follow a method-transfer protocol which defines the experiments and acceptance criteria necessary to demonstrate the analysts proficiency, equipment s suitability, and true ruggedness of the analytical method. If we assume that any quality analytical laboratory has proficient analysts who operate suitable equipment, then the method transfer stands as an ongoing means to substantiate the suitability of the original method validation. Example 5 contains an example of a method-transfer protocol for a chromatographic procedure. 

There are numerous method validation examples in the literature [9-18]. Each company has their own approach and own set of acceptance criteria for different analytical assays, but these approaches must be within the confines of their line unit QA department and be in accordance with any regulatory provisions. In the next section a description for each of the parameters to be validated (figures of merit) are described in detail and examples are given for each. 

Analysis of variance (ANOVA) tests whether one group of subjects (e.g., batch, method, laboratory, etc.) differs from the population of subjects investigated (several batches of one product different methods for the same parameter several laboratories participating in a round-robin test to validate a method, for examples see Refs. 5, 9, 21, 30. Multiple measurements are necessary to establish a benchmark variability ( within-group ) typical for the type of subject. Whenever a difference significantly exceeds this benchmark, at least two populations of subjects are involved. A graphical analogue is the Youden plot (see Fig. 2.1). An additive model is assumed for ANOVA. 

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). 

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. 

In 1994, only 15% of EPA method validations (tolerance method validation and environmental chemistry method validations) that involved GC were carried out using GC/MS. In 2002, this number is reversed in that 85% of the GC methods that were validated by both programs used GC/MS. Many of the compounds investigated in these method trials were polar compounds, and hence these compounds required derivatization in order to be amenable to GC. One common methylating agent is (trimethylsilyl)diazomethane, which is used, for example, to methylate the sulfonamide flumetsulam. As opposed to HPLC/MS, where derivatization is often not necessary, the GC/MS procedure involves an extra step to methylate this compound, under dry conditions, prior to determination by GC/MS. 

Full acceptance of HPLC/MS methods by the US EPA OPP as enforcement methods occurred between 1998 and 2001. For example, in 1998, the EPA OPP accepted HPLC/MS (without MS/MS) methods as primary enforcement methods, and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) only was suitable for confirmatory methods. However, in 2001, HPLC/MS/MS methods also became acceptable for primary enforcement. Table 4 summarizes the types of methods that were validated by the EPA OPP method validation program, for both food tolerance enforcement methods and environmental chemistry methods. 

It is well accepted that MDMA produces 5-HT depletions in rat CNS, but much less attention has been devoted to the effects of MDMA on established markers of neurotoxicity such as cell death, silver-positive staining, and reactive gliosis. Support for the hypothesis of MDMA-induced axotomy relies heavily on immunohistochemical analysis of 5-HT levels, which could produce misleading results if not validated by other methods. For example, MDMA-induced loss of 5-HT could be due to persistent adaptive changes in gene expression or protein function, reflecting a state of metabolic quiescence rather than neurotoxic damage. Table 7.3 summarizes the effects of MDMA on hallmark measures of neurotoxicity. 

One nice feature of the finite element method is the use of natural boundary conditions. It may be possible to solve the problem on a domain that is shorter than needed to reach some limiting condition (such as at an outflow boundary). The externally applied flux is still applied at the shorter domain, and the solution inside the truncated domain is still valid. Examples are given in Chang, M. W., and B. A. Finlayson, Inf. J. Num. Methods Eng. 15, 935-942 (1980), and Finla-son, B. A. (1992). The effect of this is to allow solutions in domains that are smaller, thus saving computation time and permitting the solution in semi-infinite domains. 

An indication of the minimum size of a subsample can be obtained by using the concept of a sampling constant. For example, in the laboratory, the sampling constant can be used to estimate the minimum size of the test portion. However, the suitability of the chosen test portion size must be confirmed as part of method validation. The sampling constant Ks has units of mass. This is the mass of the test portion necessary to ensure a relative subsampling error of 1% (at the 68% confidence level) in a single determination. The value of /Ks is numerically equal to the coefficient of variation, CV (see Chapter 6, Section 6.1.3) for results obtained on 1 g subsamples in a procedure with insignificant analytical error. 

You may have included some other documents with the SOPs and WIs because they are also held in your area of the laboratory. These could be, for example, guides for carrying out particular activities, such as method validation, and international guidelines on how to achieve reliable results. Some areas of activity may be covered by legislation copies of the relevant documents will be kept locally as it may be referenced in the SOP. Equipment manuals are also kept locally. 

Snee, R. D. Technometrics. 19, 1977, 415 128. Validation of regression models Methods and examples. 

This chapter focuses on approaches to the validation of high-performance liquid chromatography methods based on regulatory guidance documents and accepted industry practices. The information in this chapter gives a brief review of the reasons for performing method validation and the regulations that describe this activity. Individual validation parameters are discussed in relation to the type of method to be validated. Examples of typical validation conditions are presented with references to additional information on individual topics. This chapter was written to help analysts responsible for method validation. 

Fig. 7.14 Example for standard matrix sample. Matrix was obtained by incubation of mGATl-membrane preparation in Tris-NaCI buffer ( 10 ig protein content), subsequent filtration and elution with methanol. This empty matrix was then spiked with NO 711 and [ Hio]NO 711 to obtain standard matrix samples for LC-MS method validation purposes, (a) Trace for 0.015 nM NO 711 (mass transition 381 180, m/z). (b) Trace for 1 nM [ Hio]NO 711 (391 190, m/z) in the same sample.

Residuals A measurement residual is the part of a measurement vector that is not explained by a model (Residual = Actual Data-Data Reconstructed Using the Model). For example, when using a factor-based method to build models with spectral data, the portion of a spectrum that is not used by the model is the residual. Concentration residuals are the differences between the true (or known) and predicted concentrations. These are only available at method validation. 

The slope of the regression line will provide an idea of the sensitivity of the regression, and hence the method that is being validated. The intercept will provide an estimate of the variability of the method. For example, the ratios percent of the intercept with the variable data at nominal concentration are sometimes used to estimate the method variability. 

Execution of the method validation protocol should be carefully planned to optimize the resources and time required to complete the full validation study. For example, in the validation of an assay method, linearity and accuracy may be validated at the same time as both experiments can use the same standard solutions. A normal validation protocol should contain the following contents at a minimum ... 

An example of the minimum requirement for potency assay of the drug substance and drug product is tabulated in Table 4. Note that the postponement of intermediate precision is aligned with previous discussion that the use of early phase analytical method resides mainly in one laboratory and is used only by a very limited number of analysts. Each individual company s phased method validation procedures and processes will vary, but the overall philosophy is the same. The extent of and expectations from early phase method validation are lower than the requirements in the later stages of development. The validation exercise becomes larger and more detailed and collects a larger body of data to ensure that the method is robust and appropriate for use at the commercial site. 

However, certain fundamental concepts of cGMPs must be applied regardless of the details of the phased appropriate method validation strategy used. Examples are (1) proper documentation, (2) change control, (3) deviations, (4) equipment and utilities qualification, and (5) proper training. 

Before any method validation is started, the scope of validation must be fixed, comprising both the analytical system and the analytical requirement. A description of the analytical system includes the purpose and type of method, the type and concentration range of analyte(s) being measured, the types of material or matrices for which the method is applied, and a method protocol. On the basis of a good analysis lies a clear specification of the analytical requirement. The latter reflects the minimum fitness-for-purpose criteria or the different performance criteria the method must meet in order to solve the particular problem. For example, a minimum precision (RSD, see below) of 5% may be required or a limit of detection (LOD) of 0.1% (w/w) [2,4,15,58]. The established criteria for performance characteristics form the basis of the final acceptability of analytical data and of the validated method [58]. 

H. A. (1999), Some practical examples of method validation in the analytical laboratory, Trends Anal. Chem., 18, 584-593. 

Any or all of these conditions can be varied. To provide some guidance, intralaboratory reproducibility is used to express changes only within a laboratory, and interlaboratory reproducibility" is used to refer to the changes that occur between laboratories, for example in proficiency testing, interlaboratory method validation studies, and the like. Interlaboratory reproducibility is usually two to three times the repeatability. 

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