Method validation accuracy studies

B. Method validation includes studies of precision and accuracy of a proposed method. How would you validate precision and accuracy (Hint Review Section 5-1.)... 

Once the determinative or confirmatory method has been developed to take full advantage of the chemical properties of the analyte molecule, a study is necessary to prove that the method is valid. Criteria for method validation are outlined in guidelines from the US FDA, US EPA, and EU. A summary of the differences in regulatory requirements for method validation is provided in Table 3. The parameters addressed by all of the regulatory guidelines include accuracy, precision, sensitivity, specificity, and practicability. 

For non-compendial procedures, the performance parameters that should be determined in validation studies include specificity/selectivity, linearity, accuracy, precision (repeatability and intermediate precision), detection limit (DL), quantitation limit (QL), range, ruggedness, and robustness [6]. Other method validation information, such as the stability of analytical sample preparations, degradation/ stress studies, legible reproductions of representative instrumental output, identification and characterization of possible impurities, should be included [7], The parameters that are required to be validated depend on the type of analyses, so therefore different test methods require different validation schemes. 

Method validation is the process of proving that an analytical method is acceptable for its intended purpose. Many organizations provide a framework for performing such validations (ASTM, 2004). In general, methods for product specifications and regulatory submission must include studies on specificity, linearity, accuracy, precision, range, detection limit, and quantitation limit. 

Analytical data generated in a testing laboratory are generally used for development, release, stability, or pharmacokinetic studies. Regardless of what the data are required for, the analytical method must be able to provide reliable data. Method validation (Chapter 7) is the demonstration that an analytical procedure is suitable for its intended use. During the validation, data are collected to show that the method meets requirements for accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, and robustness. These characteristics are those recommended by the ICH and will be discussed first. 

Method validation seeks to quantify the likely accuracy of results by assessing both systematic and random effects on results. The properly related to systematic errors is the trueness, i.e. the closeness of agreement between the average value obtained from a large set of test results and an accepted reference value. The properly related to random errors is precision, i.e. the closeness of agreement between independent test results obtained under stipulated conditions. Accnracy is therefore, normally studied as tmeness and precision. 

Figure 5.1 shows the various characteristics and stages in a method validation program. For most quantitative methods of analysis, the method characteristics that require evaluation are accuracy, sensitivity, selectivity, precision and method limitations. Each of these characteristics have contributions from various effects, all of which require consideration within a method validation study. 

The basic criterion for successful validation was that a method should come within 25% of the "true value" at the 95% confidence level. To meet this criterion, the protocol for experimental testing and method validation was established with a firm statistical basis. A statistical protocol provided methods of data analysis that allowed the accuracy criterion to be evaluated with statistical parameters estimated from the laboratory test data. It also gave a means to evaluate precision and bias, independently and in combination, to determine the accuracy of sampling and analytical methods. The substances studied in the second phase of the study are summarized in Table I. 

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 ... 

The error of an analytical result is related to the (in)accuracy of an analytical method and consists of a systematic component and a random component [14]. Precision and bias studies form the basis for evaluation of the accuracy of an analytical method [18]. The accuracy of results only relates to the fitness for purpose of an analytical system assessed by method validation. Reliability of results however has to do with more than method validation alone. MU is more than just a singlefigure expression of accuracy. It covers all sources of errors which are relevant for all analyte concentration levels. MU is a key indicator of both fitness for purpose and reliability of results, binding together the ideas of fitness for purpose and quality control (QC) and thus covering the whole QA system [4,37]. 

On the one hand, even if an in-house vahdated method shows good performance and reliable accuracy, such a method cannot be adopted as a standard method. In-house validated methods need to be compared between at least eight laboratories in a collaborative trial. On the other hand, a collaborative study should not be conducted with an unoptimized method [58]. Interlaboratory studies are restricted to precision and trueness while other important performance characteristics such as specificity and LOD are not addressed [105]. For these reasons, single-laboratory validation and interlaboratory validation studies do not exclude each other but must be seen as two necessary and complementary stages in a process, presented in Figure... 

Accuracy. The accuracy of the method expresses the closeness of agreement between the experimental result and the true value. When converting a validated manual method to an automated procedure, it may not be necessary to perform an accuracy study. Instead, it may be sufficient to rely on the comparison to manual data and on additional supporting validation on the automated procedure. When developing and validating the automated method as the first-intent method, it will... 

Equivalency. This test compares the results of the automated procedure with the results of the validated manual method. If accuracy of the automated procedure has been proven, it may not be necessary to perform the equivalency study. However, if the manual method does not exist, then accuracy and reproducibility data should be used to assess the suitability of the automated method. The recommended testing for content uniformity, assays, degradation and impurity methods and dissolution methods are listed in Table 5.4. 

The fundamental parameters for bioanalytical validations include accuracy, precision, selectivity, sensitivity, reproducibility, stability of the drug in the matrix under study storage conditions, range, recovery, and response function (see Section 8.2.1). These parameters are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation, such as selectivity and quantification issues. 

The results of these interlaboratory studies are reported in USEPA Method Validation Studies 14 through 24 (14). The data were reduced to four statistical relationships related to the overall study 1, multilaboratory mean recovery for each sample 2, accuracy expressed as relative error or bias 3, multilaboratory standard deviation of the spike recovery for each sample and 4, multilaboratory relative standard deviation. In addition, single-analyst standard deviation and relative standard deviation were calculated. 

Analytical methods validation is a critical component of the entire company validation program. A method is not declared acceptable until a collaborative crossover study is conducted between two development laboratories and at least one quality control laboratory to ensure proper precision, accuracy, and efficiency. In the new world of outsourcing, it is imperative that an analytical crossover study be conducted between the client and supplier before any work is begun on dosage form development. 

The interpretation and implementation of published methods invariably differ at different laboratories due to diversity of utilized instruments, their incidental elements and supplies, and the differences in method interpretation. Each analytical method must be validated at the laboratory before it is used for sample analysis in order to demonstrate the laboratory s ability to consistently produce data of known accuracy and precision. Method validation includes the construction of a calibration curve that meets the acceptance criteria the determination of the method s accuracy and precision and the MDL study. A method SOPs must be prepared and approved for use. Method validation documentation is kept on file and should be always available to the client upon request. 

While a substitute matrix can be used to prepare standard cahbrators for a drug compound that exists endogenously, VS/QCs should be prepared in the authentic matrix, regardless. VS data are used during method validation to characterize the intra- and inter-mn accuracy/precision and stability. QC data are used for assay performance monitoring and to accept or reject a run during in-study validation. 

The filter compatibility and accuracy experiments should provide analysis values within the precision of the DS method. The specificity experiment should also show no interference from placebo components or degradants. If these criteria are met, there is a high level of confidence that method validation will prove successful. Any values outside the expected range would necessitate method modification with a subsequent feasibility study. 

Zhang et al. [43] developed and validated a stability indicating HPLC method for the determination of lornoxicam in pharmaceutical formulation. The isocratic procedure was performed in Shimadzu ODS (4.6 mm x 15 cm, 5 pm) column maintained at 25 °C. The mobile phase was degassed mixture of sodium acetate (0.05 mol/L, pH 5.8) and methanol (55 45). The flow rate was 1 ml/min and detection at 290 nm. Selectivity, specificity, linearity, precision, accuracy, and robustness were evaluated to validate the analytical method. Forced degradation studies were performed to provide an indication of the stability-indicating capacity. The stability indicating method for lornoxicam in the injectable dosage was developed and validated. The method can be considered for routine analysis and quality control of lornoxicam in injectable formulation. 

There are only a few studies dealing with method validation of the determination of environmental pollutants by CE. However, some authors have demonstrated the application of their developed separation methods. The accuracy determination for Na, K, Ca, and Mg metal ions has been presented. Similarly, the precision of migration times and peak areas for seven alkali and alkaline earth metals has been measured RSD values were less than 0.4% for migration times and from 0.8% to 1.8% for peak areas.In one of the experiments, the reported %RSD varied from 2.79 to 3.38 for Zn, Cu, and Fe metal ions. Several other studies have shown reliable results with recoveries close to 100%, or good agreement with the results obtained by other methods.In spite of this, the precision of linearity, sensitivity, and reproducibility of CE methods for metal ions and anions analysis are not better than ion chromatography. 

The first problem encountered when studying literature on method validation are the different sets of terminology employed by different authors. A detailed discussion of this problem can be found in the review of Hartmann et al. [9], Therein, it was proposed to adhere, in principle, to the terminology established by the ICH [13], except for accuracy, for which the use of a more detailed definition was recommended (cf. Accuracy). However, the ICH terminology lacked a definition for stability, which is an important parameter in bioanalytical method validation. Furthermore, the ICH definition of selectivity did not take into account interferences that might occur in bioanalysis (e.g. from metabolites). For both parameters, however, reasonable definitions were provided by Conference Report II [10]. 

Results from previous interlaboratory validation studies on the method showed accuracy to be acceptable -relative reproducibility standard deviations were between 20% and 40% depending on the hydrocarbon concentration and the amount of interfering compounds in the sample. Recoveries between 80% and 100% were... 

A new requirement of the final CLIA regulations is that laboratories must perform method validation studies oh all new tests introduced after April 24, 2003. Before this, laboratories that implemented new methods and analytical systems that had been cleared by the Food and Drug Administration (FDA) could simply follow manufacturers directions for operation and assume that the manufacturer s performance claims were valid. With the issuance of the final rule, the performance of all new tests must be validated in each laboratory to document the reportable range, precision, accuracy, and reference intervals. For some methods, it may also be necessary to determine the detection limit and to test for possible interferences. 

To avoid bias in the evaluation of the actual precision and accuracy of the bioanalytical method, the results of all QC samples assayed within accepted analytical runs should be reported and taken into consideration in the descriptive statistical analysis. Exclusion of values should be considered only in the case of a documented analytical problem (e.g. chromatographic interference) and the reason for the exclusion should be reported. This applies to both the pre-study validation of the method and the study phase itself. 

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