Specificity of an analytical method

Selectivity is often referred to as the specificity of an analytical method and is a measure of the discriminating ability of the technique. The general requirement for specificity is that the method should be capable of unambiguously determining the compounds of interest in the presence of impurities, degradation products and other sample matrix components. A specificity study often involves accelerated degradation studies to ensure all degradation products will not interfere and the collection of likely process impurities. Often a placebo sample is assayed to check for interference fi om the sample matrix. 

The specificity of an analytical method is its ability to measure accurately an analyte in the presence of interferences that are known to be present in the product synthetic precursors, excipients, enantiomers, and known (or likely) degradants that may be present. For separation techniques, this means that there is resolution of >1.5 between the analyte of interest and the interferents. 

One practical approach to1 testing the specificity of an analytical method is to compare the test results of samples containing impurities versus those not containing impurities. The bias of the test is the difference in results between the two types of samples [20]. The assumption to this approach is that all the interferents are known and available to the analyst for the spiking studies. 

Definition The specificity of an analytical method is its ability to measure, both accurately and specifically, the analyte in the presence of components that may be expected to be present in the sample matrix. Specificity may often be expressed as the degree of bias of test results obtained by analysis of samples containing added impurities, degradation products, or related chemical compounds when compared with test results from samples without added substances. The bias may be expressed as the difference in assay results between the two groups of samples. Specificity is a measure of the degree of interference (or absence thereof) in the analysis of complex sample mixtures. 

To demonstrate the validity of an analytical method, data regarding working range/ calibration, recovery, repeatability, specificity and LOQ have to be provided for each relevant sample matrix. Most often these data have to be collected from several studies, e.g., from several validation reports of the developer of the method, the independent laboratory validation or the confirmatory method trials. If the intended use of a pesticide is not restricted to one matrix type and if residues are transferred via feedstuffs to animals and finally to foodstuffs of animal origin, up to 30 sets of the quality parameters described above are necessary for each analyte of the residue definition. Table 2 can be used as a checklist to monitor the completeness of required data. 

Verification implies that the laboratory investigates trueness and precision in particular. Elements which should be included in a full validation of an analytical method are specificity, calibration curve, precision between laboratories and/or precision within laboratories, trueness, measuring range, LOD, LOQ, robustness and sensitivity. The numbers of analyses required by the NMKL standard and the criteria for the adoption of quantitative methods are summarized in Table 10. 

In 1965, Van der Grinten4 introduced the interesting concept of measureable-ness a as qualification of an analytical method in relation to the process to be regulated, with its specific fluctuation velocity. In this connection he also defined... 

It is recommended that the protocol itself contain language that allows for minor modifications of an analytical method or procedure without necessitating an amendment (or a deviation) for example, "Minor modifications in instrumental parameters and/or adjustments in technique may be made in the method during specimen analysis to enhance overall efficiency or the sensitivity, specificity, or selectivity of analyte response."... 

In the context of an analytical method to establish the accuracy, precision, reproducibility, response function and the specificity of the analytical method with reference to the biological matrix to be examined and the analyte to be quantified. 

The precision of a test method is the variability between test results obtained on the same material using a specific test method (ASTM, 2004 Patnaik, 2004). The precision of a test is usually unrelated to its accuracy. The results may be precise, but not necessarily accurate. In fact, the precision of an analytical method is the amount of scatter in the results obtained from multiple analyses of a homogeneous sample. To be meaningful, the precision study must be performed using the exact sample and standard preparation procedures that will be used in the final method. Precision is expressed as repeatability and reproducibility. 

Two somewhat different types of null hypotheses are tested, one during the development and validation of an analytical method and the other each time the method is used for one purpose or another. They are stated here in general form but they can be made suitably specific for experimentation and testing after review and specification of the physical, chemical and biochemical properties of the analyte, the matrix, and any probable interfering substances likely to be in the same matrix. Further, the null hypotheses of analytical chemistry are cast and tested in terms of electronic signal to noise ratios because modern analytical chemistry is overwhelmingly dependent on electronic instrument responses which are characterized by noise. 

Titrimetric methods are still widely used in pharmaceutical analysis because of their robustness, cheapness and capability for high precision. The only requirement of an analytical method that they lack is specificity. This chapter covers the theoretical basis of most of the commonly used methods the practical aspects of titrations have been covered thoroughly by other textbooks. 

Traceability and MU both form parts of the purpose of an analytical method. Validation plays an important role here, in the sense that it confirms the fitness-for-purpose of a particular analytical method [4]. 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 [7]. Validation is 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 [2,11]. In Section 8.2.3, the classical method validation approach is described based on the evaluation of a number of method performance parameters. Summarized, the cri-teria-based validation process consists of precision and bias studies, a check for... 

The purpose of an analytical method is the deliverance of a qualitative and/or quantitative result with an acceptable uncertainty level. Therefore, theoretically, validation boils down to measuring uncertainty . In practice, method validation is done by evaluating a series of method performance characteristics, such as precision, trueness, selectivity/specificity, linearity, operating range, recovery, LOD, limit of quantification (LOQ), sensitivity, ruggedness/robustness, and applicability. Calibration and traceability have been mentioned also as performance characteristics of a method [2, 4]. To these performance parameters, MU can be added, although MU is a key indicator for both fitness for purpose of a method and constant reliability of analytical results achieved in a laboratory (IQC). MU is a comprehensive parameter covering all sources of error and thus more than method validation alone. 

Method validation should confirm that the analytical procedure employed for a specific test is suitable for its intended use. The validation of an analytical method... 

The sensitivity and simplicity of an analytical method always improves when selective detection can be used. Because of detection selectivity, a sample in a complex matrix can be analysed with minimum sample preparation. Wouldn t it be ideal to have an ultra-specific reagent that would allow the determination of a compound in an environment containing thousands of substances ... 

Specificity is the ability of an analytical method to distinguish the analyte from everything else that might be in the sample. Electrophoresis is an analytical method in which substances... 

The same factor is used for the scale-up of the sample load. The translation of an analytical method to a preparative system is obviously dependent on the sorbent characteristics of the preparative column. Several reviews on preparative methods, column-packing techniques, theory, and equipment design have been published [132-135,145]. Specific examples of the application of preparative procedures are listed in Table 5.3. 

Procedure a specific part of an analytical method that is concerned with one aspect of the method, for example, the liquid-liquid extraction of groups of similar analytes from a water sample. 

The second approach to specificity in analysis is fortunately more readily achievable. It involves separating all the components of a mixture from one another (or at least from the analyte) before measurement takes place. To a certain extent this may be done in the sample pre-treatment step of an analytical method which, as well as ensuring the analyte is in a suitable phase in a suitable concentration for measurement, typically involves some form of sample clean-up . By using simple separation pro-... 

The selection of reference materials is therefore critical in validating the performance of an analytical method (see Chapter 1). CRMs should be used at least in the initial evaluation studies and in establishing the acceptability of calibrators used in routine service. The specific characteristics of calibrators should be documented, along with the number of different concentrations of calibrating solutions and the frequency of their use. These latter choices depend on the characteristics of tlie analytical method, particularly the stability, reproducibility, and linearity. 

The accuracy of an analytical method is defined as the inverse of the difference between an estimate and the true value that would be obtained by an ideal method. It is heavily dependent upon the specificity of the method. The precision is defined as a function of the agreement between repeated estimates carried out on fractions of the same sample, and is... 

It has been proved that the selectivity of an analytical method is directly connected to the complexity of the matrix from which the analyte must be determined. As a result, the same method can be more selective or less selective, depending on the qualitative and quantitative composition of the matrix from which the analyte must be determined. For example, two types of electrochemical sensors are described for the assay of thyroid hormones L-T3 and L-T4. The first is an amperometric biosensor based on L-amino acid oxidase (l-AAOD),270 whereas the other is an amperometric immunosensor based on anti-L-T3 and anti-L-T4.271 If T3 and T4 have to be determined from phramaceutical products, both types of sensors have the necessary sensitivity and selectivity. When it is required to determine both hormones in biological fluids or in thyroid tissue, the proposed biosensors are not selective enough because l-AAOD catalyzes the reactions of both thyroid hormones. The amperometric biosensors can only make the discrimination between the two thyroid hormones, namely, L-T3 and L-T4, since the specific antibody reacts only with the specific antigen. 

The limit of detection of an analytical method must be directly correlated with the concentration of the analytes in the sample.329 Generally, the magnitude order for the limit of detection for different types of methods (e.g., anodic stripping voltammetry, potentiometry, atomic absorption spectrometry, UV/Vis, etc.) is known. Also, the approximate concentration range of the analyte in samples is known. For validation of the method for the analysis of an analyte from a specific sample, the limit of detection must be lower than the concentration of the analyte in the sample. 

The general aspects of proving the validity of an analytical method were discussed in Section 1.4.4. In detail, the following specific recommendations for bioanalytics can be derived [32-34]. 

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