Validation of an analytical method

The most important part of a new or developed method is to demonstrate its fitness of use for its intended purpose. The main criterion of testing for acceptance of a method is through a validation report with statistical support. A method must be able to provide timely, accurate and reliable results and must be relatively easy to understand and use. Validation does not rule out all potential problems, but should correct or address the more obvious ones with warnings or notifications attached. Problems increase when additional personnel, other laboratories, or different equipment are employed to perform the method but once the initial teething problems are sorted out all laboratories should be getting almost similar results. If any adjustments to a developed method are made they must be carefully noted so that future users can be informed. If these adjustments are not reported 

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The conditions for validation of an analytical method for the determination of residues in air are the same as the requirements given in Section 4.2.5. 

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. 

According to USP 28 [1], validation of an analytical method is the process by which it is established, through the conduct of laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications. Therefore, validation is an important step in determining the reliability and reproducibility of the method because it is able to confirm that the intended method is suitable to be conducted on a particular system. 

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. 

The basic calibration as described up to here is part of the (re-)validation of an analytical method... 

Validation of an analytical method is not a single event. It is a journey with a defined itinerary and stopping places as well as a final destination. 

Full validation of an analytical method usually comprises an examination of its characteristics in interlaboratory method performance studies. However, before a method is subjected to validation by collaborative studies, the method must be validated by a single laboratory, usually by the laboratory that developed or modified this particular measurement procedure. Method validation can be described as the set of tests used to establish and document the performance characteristics of a method and against which it may be judged, thereby demonstrating that the method is fit for a particular analytical purpose. 

Barroso M, Dias M, Vieira DN, Queiroz JA, Lopez-Rivadulla M (2008) Development and validation of an analytical method for the simultaneous determination of cocaine and its main metabolite, benzoylecgonine, in human hair by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 22(20) 3320-3326. doi 10.1002/rcm.3738... 

The validity of an analytical method can be verified only by laboratory studies. Therefore, documentation of the successful completion of such studies is a basic requirement for determining whether a method is suitable for its intended applications. Appropriate documentation should accompany any proposal for new or revised compendial analytical procedures. 

Following establishment of selectivity, the FIPLC method should be validated. The validation of an analytical method is meant to demonstrate the suitability of the method to perform its intended purpose. Validation is an important... 

Bradfield, K.B. Forbes, R.A. Development and validation of an analytical method for identification of granulated nicarbazin by near infrared reflectance spectroscopy. J. Near Infrared Spectrosc. 1997, 5, 41-65. 

Development and validation of an analytical method that serves as the regulatory method for determining the withdrawal time and for assessing whether or not animal-derived products contain residue concentration in excess of the established limit (violative residues of meats and animal-derived food products]... 

Validation of an analytical method is the process by which it is established, by laboratory studies, that performance characteristics of the method meet the requirement for the intended analytical applications [5],... 

Table 1 Validation of an analytical method, showing tasks and typical acceptance criteria for the analyte...

The development of a method for preparative HP-RPC purification for the purpose of isolation of one or more component(s) from a natural product sample (or alternatively the purification of a synthesized product from natural occurring precursors) is usually performed in four steps (1) development, optimization, and validation of an analytical method, (2) scaling up of this method to a preparative chromatographic system, (3) application of the preparative method to the fractionation of the product, and (4) analysis of the individual fractions. 

Cullere, L., Cacho, J., and Ferreira, V. (2006). Validation of an analytical method for the solid phase extraction, in cartridge derivatization and subsequent gas chromatographic-ion trap tandem mass spectrometric determination of l-octen-3-one in wines at ngL 1 level, Anal. Chim. Acta The Netherlands, 563(1-2), 51-57. 

In this chapter, we have covered the way s to understand, estimate, and interpret various criteria required for assessing the validity of an analytical method. Whatever the complexity or simplicity of computations and models needed, the primary obj ective of an analytical method should never be forgotten Can each measurement be trusted or, equivalently, is the measurement error acceptable All the information needed to make a decision is contained in the measurement error profile. The key performance characteristics such as the linearity, accuracy, precision, limits of quantification, and sensitivity are readily obtained from this profile and can easily be understood and interpreted by an analyst. 

A high sensitivity of the method creates low selectivity. The interrelationship between selectivity and sensitivity must be a strong consideration in the validation of an analytical method. As was shown in Chapter 9, the best quality and reliability for analytical information is obtained only when the sensitivity is at a medium level and the selectivity is high. 

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]. 

Screening in a pharmaceutical process - extrusion-spheronization Validation of an analytical method - dissolution test... 

Validation of an analytical method is often done under the best of conditions such as use of a new column on dedicated equipment by an analyst experienced with the method. But what about routine analysis of commercial samples by many analysts in a busy Quality Control laboratory Robustness establishes the reliability of the method with respect to deliberate variations in the operating parameters, evaluates use of different column lots from the vendor, and also determines the stability of sample and standard solutions. Quality by Design (QbD) principles have begun to impact method development and validation activities to a wider extent and application of QbD concepts should result in higher quality and more robust analytical... 

Wortmarm AC, Froehlich PE, Pinto RB, et al. Hep>atic iron quantification by atomic absorption spectrophotometry Full validation of an analytical method using a fast sample preparation. Spectroscopy 2007 21 161-167. 

Validation of an analytical method establishes in laboratory studies that the performance characteristics of the method meet the requirements for the intended application, thus the method does what it is expected to do. The following items are listed by ICH precision, accuracy, limit of detection, limit of quantification, specificity, range, linearity, and ruggedness. Of these, accuracy is probably the most difficult to document or obtain, at least for solid formulations. This has to do with the fact that recovery experiments are difficult to design in such a way that they resemble the process conditions. The reactions there can create interactions that are not obtained in an experiment where the analyte has only been mixed or spiked to the sample. 

Elucidation of how the general principles underlying the concept of validation should be expressed in practice is an evolving process, as exemplified by the ongoing evolution of validation requirements for bioanalytical assays in the pharmaceutical industry (Shah 1992, 2000 FDA 2001 Viswanathan 2007). The complementary principle of fitness for purpose (Section 9.2) applies not only to the assay method but also to the validation process itself. Procedures that are considered to be fit for purpose in validation of an analytical method to be used in drug development, for example, need not necessarily apply to, e.g., methods used to screen pesticide residues in foodstuffs. As noted in Section 9.2, this point of view appears to be consistent with the definition of validation applied to all measurements (ISO 1994) Validation Confirmation by examination and provision of objective evidence that the particular requirements for a specified intended use are fulfilled. Of course, some basic principles are common to all validation schemes. 

The second example of development and validation of an analytical method for pharmacokinetic studies of a candidate compound in drug development (clinical studies) concerns an additional complication rather different from the enantiomeric distinction discussed above. In this case (Sennbro 2006) the drug candidate (laquinimod) was a synthetic compound intended for oral treatment of multiple sclerosis. In pre-clinical studies the pharmacokinetics in animal models had been determined using... 

The validation of an analytical method for the determination of plasticisers (DEHA, DEHP) in PVC has been reported [90], as well as that of the calibration procedure in AAS methods [91], In an original development Van Every el al. [13] have applied infrared principle components/Mahalanobis distance discriminant (PMD) analysis to validate polyolefin (film) products. PMD is a technique designed to classify complex materials into groups or identify unknowns by using n principle components to map data characteristics into an n-space cluster [92]. An unknown material can then be assigned a distance from this cluster based on the number of standard... 

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