Pharmaceutical assays

Methodology-Theory-Instrumentation Pharmaceutical Assays-Cognate Assays... 

Traditionally, most pharmaceutical assays are isocratic analysis employing the same mobile phase throughout the elution of the sample. Isocratic analyses are particnlarly common in quality control applications since they nse simpler HPLC eqnipment and premixed mobile phases. Notable disadvantages of isocratic analysis are limited peak capacity (the maximnm nnmber of peaks that can be accommodated in the chromatogram), and problems with samples containing analytes of diverse polarities. Also, late eluters (such as dimers) are particularly difficult to quantitate in isocratic analysis due to excessive band broadening with long retention times. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

Since its creation around 1973, modern high-pressure liquid chromatography (HPLC) has played a dominant role in the analysis of pharmaceuticals. It is used in many different applications for example, in content uniformity assays and stability-indicating methods, for the purity profiles of drug substances, or in the analysis of drug metabolism in animals and humans. The heart of all of these assays is the HPLC column. In this chapter, we will describe the fundamental properties of HPLC columns as well as how these properties influence column performance and separation characteristics in pharmaceutical assays. 

HPLC precision is critical in pharmaceutical analysis.For most pharmaceutical assays under a good manufacturing practice (GMP) environment, retention time and peak area precision of <2.0% RSD must be demonstrated before any samples can be analyzed. This section reviews the fundamental principles of HPLC precision and offers practical guidelines for its enhancement. The reader is referred to Reference 18 for a more detailed treatment of this topic. 

For those scientists who had to perform quantitation, the linearity of the A/D was also critical. Linearity is the condition in which the detector s response is directly proportional to the concentration or amount of a component over a specified range of component concentrations or amounts. It is imperative that the A/D not add any additional error or variability to the performance of the detector. The resulting calibration curve now becomes dependent on the combined linearity of the detector and the /VD. Accurate quantitation requires that the system is linear over the range of actual sample concentrations or amounts. Many pharmaceutical assays, like degradation and stability studies, require that the system be able to identify and quantitate very disparate levels of peaks. In many cases, this translates into a 3 to 4 order of magnitude difference between the main active component and the impurities that need to be quantitated. 

Corti, P. Dreassi, E. Corbini, G. Lonardi, S. Viviani, E. Mosconi, L. Bemuzzi, M. Application of near infrared reflectance to the analytical control of pharmaceuticals assay of ranitidine chlorhydrate and water content in tablets. Pharm. Acta Helv. 1990, 65, 28-32. 

For pharmaceutical analyses, it is necessary to have reliable methods. Results obtained using ion-selective membrane electrodes are the best because of the simplicity, rapidity, and accuracy of direct and continuous measurement of the activity of the ions in the solution. Another very important reason for the selection of electrochemical sensors for pharmaceutical assay is non-interference of by-products when the purity of a raw material is to be determined. 

These oils, if they are to be used parenterally, need to be chemically pure and free from microbial contamination. As stated above, plant oils are often complex mixtures of chemically similar compounds and so require special forms of pharmaceutical assay (e.g. determination of their acid and... 

Repeatability is a measure of the ability of the method to generate similar results for multiple preparations of the same homogeneous sample by one analyst using the same instrument in a short time duration (e.g., on the same day). For instance, method repeatability for pharmaceutical assays may be measured by making six sample determinations at 100% concentration, or by preparing three samples at 80,100, and 120% concentration levels each. 

As with GC, HPLC can be used qualitatively for identification or quantitatively to determine how much of a compound is present. Some examples of the use of HPLC in qualitative work include identification of impurities and toxicity screening. Some examples of the use of HPLC in quantitative work are drug testing in athletes and in sports supplements, pharmacokinetic studies of drugs pharmaceutical assays and fatty acid analysis. ... 

Assay of pharmaceutical substances and formulated products is one of the most important and regulated activities in the pharmaceutical analysts laboratory. Regulatory authorities require strict validation standards to show that analytical assay methods are robust, accurate, repeatable, and suitable for their intended purpose. In the pharmaceutical industry, HPLC has dominated most analytical assay determinations and it is well established as the method of choice in most laboratories. In addition, pharmacopoeia monographs specify HPLC and titrimetric methods for the majority of pharmaceutical assays. To date, CE is not used extensively in QC work despite displaying excellent efficiencies, resolution, asymmetry factors, and signal-to-noise ratio. This is mainly due to the fact that CE can suffer from insufficient sensitivity and repeatability to control impurities in pharmaceutical substances at the levels required. These issues have been addressed somewhat with sensitivity... 

A number of validated CE methods for pharmaceutical assay have been reported a selection of recent methods along with the mode of CE used and the run buffer are shown in Table 4.3. 

Eurachem guide [285], which discusses when, why, and how methods should be validated. However, for the pharmaceutical industry, the main reference source is the ICH Guidelines [286], which provides recommendations on the various characteristics to be tested for the most common types of analytical procedures developed in a pharmaceutical laboratory. The main characteristics of any analytical method to be tested are specificity, linearity, accuracy, precision, solution stability, limits of detection and quantification, and robustness. Specific aspects should be considered for a CE method including method transfer between instrument manufacturers, reagent purity and source, electrolyte stability, capillary treatment and variations in new capillaries, and buffer depletion. Fabre and Altria [284] discuss CE method validation in more detail and include a number of examples of validated CE methods for pharmaceutical analysis. Included in Table 4.3 are a number of validated pharmaceutical assay methods. 

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