Accuracy method validation protocol

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 accuracy of the method was evaluated by assaying six independently prepared solutions of IB-367 against two standard solutions of the same lot as external standards. The mean of 102.3% met the criteria set in validation protocol (97 to 103%). 

While methods validation and accuracy testing considerations presented here have been frequently discussed in the literature, they have been included here to emphasize their importance in the design of a total quality control protocol. The Youden two sample quality control scheme has been adapted for continuous analytical performance surveillance. Methods for graphical display of systematic and random error patterns have been presented with simulated performance data. Daily examination of the T, D, and Q quality control plots may be used to assess analytical performance. Once identified, patterns in the quality control plots can be used to assist in the diagnosis of a problem. Patterns of behavior in the systematic error contribution are more frequent and easy to diagnose. However, pattern complications in both error domains are observed and simultaneous events in both T and D plots can help to isolate the problems. Point-by-point comparisons of T and D plots should be made daily (immediately after the data are generated). Early detection of abnormal behavior reduces the possibility that large numbers of samples will require reanalysis. 

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. 

The ISO definition of validation is confirmation by examination and provision of objective evidence that the particular requirements of a specified intended use are fulfilled [15]. Method validation is needed to confirm the fitness for purpose of a particular analytical method, that is, to demonstrate that a defined method protocol, applicable to a specified type of test material and to a defined concentration rate of the analyte —the whole is called the analytical system — is fit for a particular analytical purpose [4]. This analytical purpose reflects the achievement of analytical results with an acceptable standard of accuracy. An analytical result must always be accompanied by an uncertainty statement, which determines the interpretation of the result (Figure 6). In other words, the interpretation and use of any measurement fully depend on the uncertainty (at a stated level of confidence) associated with it [8]. Validation is thus the tool used to demonstrate that a specific analytical method actually measures what it is intended to measure and thus is suitable for its intended purpose [11,55,56]. 

Quantitation limit is defined in the ICH Q2A guideline as the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy [2]. As with the detection limit, ICH guideline Q2B expanded upon this statement and listed the same three approaches to the testing methodology [3]. Again the method for the determination must be listed in the validation protocol and supported by the validation experiments. 

Ideally, the expected masses are obtained by an alternative physical methodology that is highly accurate and precise. In the case of using a commercial standard, the vendor should provide statistics on the accuracy and precision of the method used to obtain/measure the stated label concentration. When using a previously released lot, the expected masses will be based on the certificate of analysis concentration for the lot of Analyte B used in this validation. In the latter case, the measurement of accuracy is relative to the historical certificate of analysis value. The validation protocol needs to state that the accuracy measurement is relative to the historical value rather than to an independently obtained measurement. 

Once a new analytical method has been developed, it must be optimized and standardized to meet the purposes for which it was developed. Validation studies confirm that a new assay has met its required performance specifications, and must be done before the analytical procedure can become a routine laboratory protocol. There are multiple approaches to validating analytical methods. The most common is to compare results from the new method with results obtained using an established, or reference, assay. Alternatively, standard reference materials (e.g. standards certified by NIST) can be analyzed by the new method to demonstrate its accuracy. When available, authentic specimens should be tested and compared against a previously validated protocol. 

As an analytical approach to residue analysis, immunoassay methods are not well characterized, and no validation protocols have been established. The Association of Official Analytical Chemists, whose primary purpose is validation of analytical methods, established a Task Force on Test Kits and Proprietary Methods (2), which has addressed some of the issues relating to immunoassay methods. The International Union of Pure and Applied Chemistry s Commission on Food Chemistry has established a Working Group on Immunochemical Methods, whose first project is to develop draft guidelines on criteria for evaluation, validation, and quality control for r o-immunoassay methods (10). Similar guidelines for EIAs will also be developed. These documents will assist in development and standardization of requirements for precision for both between-laboratories and within-laboratory andyses, accuracy, and ruggedness, and— for qualitative methods— false positive and false negative rates. 

Prior to its use a method has to be validated. Validation is the formal proof that the method is suitable for the intended purpose. This requires that all steps and parameters of the method have been clearly specified in a written method description, any necessary equipment was qualified, and acceptance criteria for each validation point have been agreed upon. For quantitative methods the International Conference on Harmonization (ICH) has issued specific guidelines for setting up a validation protocol and for parameters that have to be validated for different applications. These include specificity, accuracy, precision, LOD, LOQ, linearity, and range as well as robustness. The only required validation parameter for qualitative methods is specificity. 

Table 6.1 displays recommended specifications for wavelength accuracy, photometric linearity, and spectrophotometric noise levels for pharmaceutical applications. The first step in validating any NIR method is to test the suitability of these specifications for a given application. Wavelength accuracy tests conducted using appropriate external standards will prevent potential problems that could occur with proprietary internal calibration protocols. The exact nature of any calibration standard must be noted in a validation protocol. Rare earth oxides and glass standards are candidates for such calibration. 

Once our methods have been fully developed, and procedures drafted, a protocol driven, ICH compliant method validation should be conducted. This validation should be appropriate for this early stage of development. Typically, since only one lab is running this method, and likely one chemist, intermediate precision and robustness do not need to be evaluated. Other critical attributes, such as specificity, linearity, and accuracy will need to be evaluated. 

Whilst this Chapter is primarily concerned with the methods of determining the free energies of tautomeric or ionisation equilibria via computer simulation of free energy differences, many of the issues raised relate also to the determination of other molecular properties upon which behaviour of the molecule within the body may depend, such as the redox potential or the partition coefficient.6 In the next section, we shall give a brief explanation of the methods used to calculate these free energy differences -namely the use of a thermodynamic cycle in conjunction with ab initio and free energy perturbation (FEP) methods. This enables an explicit representation of the solvent environment to be used. In depth descriptions of the various simulation protocols, or the accuracy limiting factors of the simulations and methods of validation, have not been included. These are... 

The ability to provide accurate and reliable data is central to the role of analytical chemists, not only in areas like the development and manufacture of drugs, food control or drinking water analysis, but also in the field of environmental chemistry, where there is an increasing need for certified laboratories (ISO 9000 standards). The quality of analytical data is a key factor in successfully identifying and monitoring contamination of environmental compartments. In this context, a large collection of methods applied to the routine analysis of prime environmental pollutants has been developed and validated, and adapted in nationally or internationally harmonised protocols (DIN, EPA). Information on method performance generally provides data on specificity, accuracy, precision (repeatability and reproducibility), limit of detection, sensitivity, applicability and practicability, as appropriate. 

The key elements of an inspection are to ensure that the facility is capable of fulfilling the application commitments to manufacture, process, control, package, and label a drug product following GMP the adequacy and accuracy of analytical methods submitted, to ensure that these methods are proper for the testing proposed correlation between the manufacturing process for clinical trial material, bioavailability study material, and stability studies and submitted process that the scientific data support full-scale production procedures and controls that only factual data have been submitted and that the protocols are in place to validate the manufacturing process. 

The ideal validated method would be the one that has progressed fully through a collaborative study in accordance with international protocols for the design, conduct, and interpretation of method performance studies. A typical study of a determinative method conducted in accordance with the internationally harmonized International Organization for Standardization (ISO)/International Union for Pure and Applied Chemistry (IUPAC)/AOAC International (AOAC) protocol would require a minimum of up to five test materials including blind replicates or split-level samples to assess within-laboratory repeatability parameters, and eight participating laboratories (15). Included with the intended use should be recommended performance criteria for accuracy, precision and recovery. 

The following principles should be used to establish a valid analytical method A specific detailed description and protocol should be written (standard operating procedure (SOP)). Each step in the method should be investigated to determine the extent to which environmental, matrix, material, or procedural variables, from time of collection of material until the time of analysis and including the time of analysis, may affect the estimation of analy te in the matrix. A method should be validated for its intended use with an acceptable protocol. Wherever possible, tire same matrix should be used for validation purposes. The concentration range over which the analyte will be determined must be defined in the method, on the basis of actual standard samples over the range (standard curve). It is necessary to use a sufficient number of standards to adequately define the relationship between concentration and response. Determination of accuracy and precision should he made by analysis of replicate sets of analyte samples of known concentration from equivalent matrix. 

Item b. The method was changed to allow for gravimetric sample preparation. A revalidation experiment will be performed as per a QAU-approved protocol. This revalidation experiment will repeat the accuracy and precision sections of the original validation. The acceptance criteria will be the same as for the original validation. 

Finally a validation step for the overall MISPE procedure is mandatory to allow the use of the method itself in place of the regulatory methods. The issues to be considered here are the accuracy and precision of the method based on the MISPE protocol, its limit of quantification, selectivity and ruggedness. In particular the inter- and intra-assay precision need to be checked with real samples and certified reference materials and methods [23,24]. 

Regarding each of the above three methods, DPRA, Keratin oSens, and h-CLAT, all of them have well defined protocols and prediction models and each assay has been tested with >100 substances, achieving a predictive accuracy of approximately 80 %. It is for this reason that they have been able to enter the formal validation process [46, 59],... 

Accuracy is a measure of how close to truth a method is in its measurement of a product parameter. In statistical terms, accuracy measures the bias of the method relative to a standard. As accuracy is a relative measurement, we need a definition of true or expected value. Often, there is no gold standard or independent measurement of the product parameter. Then, it may be appropriate to use a historical measurement of the same sample or a within-method control for comparison. This must be accounted for in the design of experiments to be conducted for the validation and spelled out in the protocol. Accuracy is measured by the observed value of the method relative to an expected value for that observation. Accuracy in percent can be calculated as ratio of observed to expected results or as a bias of the ratio of the difference between observed and expected to the expected result. For example, suppose that a standard one-pound brick of gold is measured on a scale 10 times and the average of these 10 weights is 9.99 lbs. Then calculating accuracy as a ratio, the accuracy of the scale can be estimated at (9.99/10) x 100% = 99.90%. Calculating the accuracy as a bias then [(9.99 - 10)/10] X 100% =-0.10% is the estimated bias. In the first approach ideal accuracy is 100%, and in the second calculation ideal bias is 0%. 

For all these reasons, TVOC data from the literature must be interpreted very cautiously and it would be beneficial to have a standardized method for reporting TVOC. This should include specific sampling and analytical protocol and should provide some validation of the methods used, including a specification of the simplifications made and limitations of the accuracy following from these simplifications. 

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