Validation, method precision

Key words Measurement uncertainty Method validation Precision Trueness Ruggedness testing... 

Precision, which quantifies the variation between replicated measurements on test portions from the same sample material, is also an important consideration in determining when a residue in a sample should be considered to exceed a MRL or other regulatory action limit. Precision of a method is usually expressed in terms of the within-laboratory variation (repeatability) and the between-laboratory variability (reproducibility) when the method has been subjected to a multi-laboratory trial. For a single-laboratory method validation, precision should be determined from experiments conducted on different days, using a minimum of six different tissue pools, different reagent batches, preferably different equipment, and so on, and preferably by different analysts Repeatability of results when determined within a single laboratory but based on results from multiple analysts is termed intermediate precision Precision of a method is usually expressed as the standard deviation. Another useful term is relative standard deviation, or coefficient of variation (the standard deviation divided by the absolute value of the arithmetic mean result, multiplied by 100 and expressed as a percentage). 

The last step in establishing a standard method is to validate its transferability to other laboratories. An important step in the process of validating a method is collaborative testing, in which a common set of samples is analyzed by different laboratories. In a well-designed collaborative test, it is possible to establish limits for the method s precision and accuracy. 

During the method validation phase, the calibration, using the CS solutions, is repeated each day over at least one week to establish both the within-day and the day-to-day components of the variability. To this end, at least 6 CS, evenly spread over the concentration range, must be repeatedly run (m = 8-10 is usual), to yield n 50 measurements per day. If there are no problems with linearity and heteroscedacity, and if the precision is high (say, CV < 2-5%, depending on the context), the number of repeats m per concentration may be reduced from the second day onwards (m = 2 - 3 is reasonable). The reasoning behind... 

In this article, an analytical method is defined as series of procedures from receipt of a sample to final determination of the residue. Validation is the process of verifying that a method is fit for purpose. Typically, validation follows completion of the development of a method. Validated analytical data are essential for monitoring of pesticide residues and control of legal residue limits. Analysts must provide information to demonstrate that a method intended for these purposes is capable of providing adequate specificity, accuracy and precision, at relevant analyte concentrations and in all matrices analyzed. 

Most often studies will be accepted by regulatory authorities even if they do not contain all information. For example, a summary, the scope, a separate notice regarding the residue definition or a schematic diagram of the analytical procedure are helpful and may avoid additional questions, but they are not essential. Also, detailed specification of standard glassware or chemicals commonly used in residue analysis is less important. Finally, data about extraction efficiency or analyte stability can be offered in separate studies or statements, which are also valid for other methods. However, each method must precisely describe at the minimum ... 

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. 

RACs. The LOD and LOQ for each study should be determined. Until each study s LOD and LOQ have been determined, use the lower limit of method validation (LLMV) of 0.01 mgkg for each metabolite class as the reference LOQ. LLMV is defined as the lowest level of fortification where we have demonstrated acceptable recovery and precision of EMA- and HEMA-producing metabolites. 

Once you have confidence that your method is adequate from the preliminary work in the method tryout, you are ready to begin the method validation. The method validation provides additional data on accuracy and precision, and confirms that there are no problems due to interference. Method validation must be completed before beginning the analysis of the treated samples from the field. The validation should test the detector s response over the expected range of concentrations from the field. 

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 defined in the international standard, ISO/IEC 17025 as, the confirmation by examination and provision of objective evidence that the particular requirements for a specific intended use are fulfilled. This means that a validated method, if used correctly, will produce results that will be suitable for the person making decisions based on them. This requires a detailed understanding of why the results are required and the quality of the result needed, i.e. its uncertainty. This is what determines the values that have to be achieved for the performance parameters. Method validation is a planned set of experiments to determine these values. The method performance parameters that are typically studied during method validation are selectivity, precision, bias, linearity working range, limit of detection, limit of quantitation, calibration and ruggedness. The validation process is illustrated in Figure 4.2. 

Precision estimates are key method performance parameters and are also required in order to carry out other aspects of method validation, such as bias and ruggedness studies. Precision is also a component of measurement uncertainty, as detailed in Chapter 6. The statistics that are applied refer to random variation and therefore it is important that the measurements are made to comply with this requirement, e.g. if change of precision with concentration is being investigated, the samples should be measured in a random order. 

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. 

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. 

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. 

To support the development activities, accurate and sensitive analytical methods are required. With progress in development, increasing method validation work is performed and the acceptance criteria for drug quality and test method precision are tightened. 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

Reproducibility represents the precision of the method between two or more laboratories and it is typically assessed during method transfer between laboratories, but may be assessed during method validation when more than one laboratory will be performing the method. Reproducibility would also be reported as the SD or RSD value of the mean results between laboratories. These data are not part of the marketing authorization application. 

Resorcinol Water solution Non-destructive quantitation method. Precision lower than 0.15% in a temperature range of 9-35 °C. Method validated using ICH-adapted guidelines 151... 

Step 5 Off-line method or analyzer development and validation This step is simply standard analytical chemistry method development. For an analyzer that is to be used off-line, the method development work is generally done in an R D or analytical lab and then the analyzer is moved to where it will be used (QA/ QC lab, at-line manufacturing lab, etc.). For an analyzer that is to be used on-line, it may be possible to calibrate the analyzer off-line in a lab, or in situ in a lab reactor or a semiworks unit, and then move the analyzer to its on-line process location. Often, however, the on-line analyzer will need to be calibrated (or recalibrated) once it is in place (see Step 7). Off-line method development and validation generally includes method development and optimization, identification of appropriate check samples, method validation, and written documentation. Again, the form of the documentation (often called the method or the procedure ) is company-specific, but it typically includes principles behind the method, equipment needed, safety precautions, procedure steps, and validation results (method accuracy, precision, etc.). It is also useful to document here which approaches did not work, for the benefit of future workers. 

Accuracy expresses the closeness of a result to the true value. Accuracy = trueness + precision. Under specific conditions it is quantified by the measurement uncertainty. Measurement uncertainty may vary under changing conditions and method validation determines the degree. 

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. 

A ruggedness test is a part of method validation (Table 3.1) and can be considered as a part of the precision evaluation [2,4,5]. Ruggedness is related to repeatability and reproducibility. Some definitions for ruggedness come very close to those for reproducibility. Certain interpretation methods to identify the significant factors in a ruggedness test use criteria based on results for repeatability or reproducibility. These two items will be considered in Section 3.4.7. 

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 study of the precision of a method is often the most time and resource consuming part of a method validation program, particularly for methods that are developed for multiple users. The precision is a measure of the random bias of the method. It has contributions fi om the repeatability of various steps in the analytical method, such as sample preparation and sample injection for HPLC [5-9], and from reproducibility of the whole analytical method fiom analyst to analyst, fiom instrument to instrument and fiom laboratory to laboratory. As a reproducibility study requires a large commitment of time and resources it is reasonable to ensure the overall ruggedness of the method before it is embarked upon. 

There are several reasons for careful placement of the ruggedness test in a program of method validation tests. Firstly the ruggedness test itself can be a complex and time consuming task and thus should be carried out as late in the method validation as possible, (i.e. when most other performance characteristics have been established and are acceptable). This reduces the chance of a failed ruggedness test and for this reason it is recommended that the precision study be one of the last experiments in a validation study. 

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