Linearity and Range

The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in a sample within a given range [81], Linearity should be evaluated in the concentration span 80-120 % of the expected con- 

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Accuracy and precision Specificity Limit of detection Limit of quantitation Linearity and range... 

Linearity and range Linearity is an inherent property of NMR spectroscopy. A standard curve can be easily obtained to cover a wide range of concentrations with a typical R2 value of >0.99. 

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. 

This chapter deals with the validation of capillary electrophoresis (CE) methods. It describes the various validation characteristics, namely accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range in accordance with the official guidelines. Practical aspects related to the calculation of these parameters and factors affecting them in CE analysis have also been described. Validation requirements have been described according to the goal of the method. The chapter contains numerous tables and diagrams to illustrate these ideas. It also covers other related aspects such as instrument qualification, revalidation, and method transfer. 

This chapter sheds light on the different validation requirements and methods to investigate them. Evaluation of the typical validation characteristics, namely accuracy, precision, specificity, DL, QL, linearity, and range in CE, has been discussed in details. Validation in CE is similar to validation in other separation techniques such as HPEC, but in CE, the capillary surface properties and namely the EOF have to be especially addressed. Eurther, the instrument performance has to be carefully considered during validation and method transfer. Here, the condition of the lamp and the thermostating system is of particular importance. 

For a method to be considered as part of commercial specification, validation using ICH guidelines (Q2A and Q2B) is required (see also Chapters 9 and 10). Depending on method characteristics, different validation schemes may be used. The following parameters should be considered to ensure the method is valid and appropriate for its intended purpose accuracy, precision (repeatability, intermediate precision), specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, and range. 

Table 5 summarizes the comparison of the vahdation requirements with the verification requirements of the HPLC assay of an example final dosage form. ICH requires the validation of accuracy, precision, specificity, linearity, and range. Generally, verification will only require a minimal of precision and specificity validation. The accuracy requirements will be dependent on the specific situation of the final dosage form. 

ICH Q2A suggested validation of the characteristics of accuracy, precision, specificity, linearity, and range for potency and content uniformity assay. A detailed discussion of each of these parameters is presented later in this chapter. Some examples of validation data are presented along with a brief critical discussion of the data. 

Limits of detection and quantitation Linearity and range Ruggedness Robustness... 

For proteins, most applications rely on immunoassays and alternatives are not readily available total recovery and possible denaturation that renders the extracted proteins undetectable by antibodies are the most critical factors. Depending on the tissue type analyzed (leaves, seeds, roots) validation needs to be performed separately on all different tissue types. For each tissue type the extraction efficiency as well as sensitivity, linearity and range of the method (amongst other parameters) need to be assessed. 

Standard Preparation into the substrate, and mix well. Record the decrease in absorbance over 3 min, recording the absorbance value approximately every 15 s. The rate of the decrease in absorbance should be linear, and range between 0.03 and 0.06 per min. Repeat the procedure with the Sample Preparation. 

Determination of Linearity and Range Determine the linearity of an analytical method by mathematically treating test results obtained from analysis of samples with analyte concentrations across the claimed range of the method. The treatment is normally a calculation of a regression line by the method of least squares of test results versus analyte concentrations. In some cases, to obtain proportionality between assays and sample concentrations, the test data may have to be subjected to a mathematical transformation before the regression analysis. The slope of the regression line and its variance (correlation coefficient) provide a mathematical measure of linearity the y-intercept is a measure of the potential assay bias. 

The United States Pharmacopoeia (U.S.P.) [5] in a chapter on validation of compendial methods, defines analytical performance parameters (accuracy, precision, specificity, limit of detection, limit of quantitation, linearity and range, ruggedness, and robustness) that are to be used for validating analytical methods. A proposed United States Pharmacopeia (U.S.P.) general chapter on near-infrared spectrophotometry [6] addresses the suitability of instrumentation for use in a particular method through a discussion of operational qualifications and performance verifications. 

Finally, McGonigle discusses the types of method modifications that require revalidation. Any modification that could potentially affect the accuracy, precision, linearity, and range of a method require revalidation of these parameters. 

The FDA has identified seven validation characteristics accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range. Depending on the test being validated, combinations of these characteristics need to be examined. 

This guideline refers to terms and definitions of parameters included in validation experiments, whereas Q2B describes the way in which validation can be performed. Attributes covered in Q2A include specificity (for identification tests) accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range (for impurity tests) and accuracy, precision, specificity, linearity, and range for assay measurements (e.g., content, potency, and dissolution testing). 

Demonstration of Linearity and Range Determination of Relative Response Factor... 

To validate SOP 123 for measuring mass of Product W, the quantitative method performance characteristics of accuracy, precision, linearity, and range will be assessed using the validation assays shown in the design matrix over two days and using two operators. As per ICH guideline Q2A, the validation experiments will be run from 40 pg to 180 jUg. The test lot will be diluted out and concentrated up to specific expected masses using the mass cited on certificate of analysis for the lot of Product W selected for the validation. The points on the Product W curve will be as follows 40 /xg, 50 /xg, 70 /xg, 90 /xg, 110 /xg, 130 pg, 150 pg, and 180 pg. 

Linearity and range define the range in which, by calibration, a direct proportionality of analytical detection signal and concentration or potency is given, taking into account sufficient precision and accuracy of results. 

Linearity and range. The linearity of an analytical procedure is its ability to produce results that are directly proportional to the concentration of analyte in the samples. The range of the procedure is an expression of the lowest and highest levels of analyte that have been demonstrated to be determinable with acceptable precision, accuracy, and linearity. These characteristics are determined by application of the procedure to a series of samples having analyte concentrations spanning the claimed range of the procedure. When the relationship between response and concentration is not linear, standardization may be provided by means of a calibration curve. 

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. 

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