Experiments for Validation of Quantitative Methods

To understand why HPLC is frequently used in quantitative analytical methods, it is useful to assess whether or not an analytical method is suitable for its intended purpose and, in doing so, consider the deficiencies in methods employing classical measurement steps. Analytical method validation is the process of assessing the fitness for purpose of an analytical method in choosing an analytical method issues such as cost, simphcity, operator experience, availability etc. are of secondary importance to the actual validity of the method under consideration. In the validation procedure, tests are typically carried out for the following properties ... 

With this rapid growth of near-infrared spectroscopic research in the health sciences, it is time for a text such as this. The authors have combined more than 35 years of industrial and university research experience in this volume. The pharmaceutical presentation is arranged in a logical progression theory, instrumentation, physical manipulation (blending, drying, and coating), analysis (both qualitative and quantitative), and finally, validation of the method. The varied mathematics used in NIR, called chemometrics, are only briefly mentioned. Detailed explanations and applications are covered in texts or chapters devoted to the subject [1-4]. 

The next section deals with method validation of quantitative TLC methods. Two questions should, however, be answered prior to discussing the validation experiments namely, whether the statistical evaluation of data elements, such as precision, accuracy, and reproducibility should be calculated on the basis of measured peak heights or peak areas, and whether the internal or external standard methods, or area normalization should be used to yield quantitative results for the assay. Without going into detail, the most important advantages and limitations of peak height and peak area measurements, and those of the different methods of quantification are summarized in Table 4. 

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. 

The quantitative assay for PBG and ALA (Bio Rad, Hercules, CA, USA) that is based on the classical method by Mauzerall and Granick may be used for determination of the porphyrin precursors. PBG is absorbed by the anion-exchange column and ALA by the cation-exchange column interferences are washed out. After elution from the column, ALA is derivatized by acetyl acetone to form a pyrrole. Both ALA and PBG are determined colorimetrically with the modified Ehrlichs reagent. Instead of this broadly used standard method ALA, but not PBG may be detected and quantified using amino acid chromatography. However, our experience has shown that this method is only valid for detecting massively increased concentrations of ALA. 

In terms of traditional quantitative validation, the most efficient, albeit slowest, approach was to validate by disease and metabolites. It was also helpful to focus on key metabolites in disease and not every marker potentially detectable from a quantitative perspective. Other metabolites that did not have a standard could be considered supportive qualitative markers. This approach could then be incorporated into a method for interpretation, a process that continues to refine itself based on the screening experience for rare disorders, method improvement, and secondary or confirmatory test. 

Heterogeneously catalyzed reactions are usually studied under steady-state conditions. There are some disadvantages to this method. Kinetic equations found in steady-state experiments may be inappropriate for a quantitative description of the dynamic reactor behavior with a characteristic time of the order of or lower than the chemical response time (l/kA for a first-order reaction). For rapid transient processes the relationship between the concentrations in the fluid and solid phases is different from those in the steady-state, due to the finite rate of the adsorption-desorption processes. A second disadvantage is that these experiments do not provide information on adsorption-desorption processes and on the formation of intermediates on the surface, which is needed for the validation of kinetic models. For complex reaction systems, where a large number of rival reaction models and potential model candidates exist, this give rise to difficulties in model discrimination. 

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