Assay methods robustness

As an identity (ID) test, per ICH guidelines, only selectivity is required in method qualification and validation. Repeatability and intermediate precision are often included to ensure reliability of p7 determinations. Additionally, method robustness should be tested to assure that the assay performance is suitable for QC environment. Quantitative parameters such as LOD/LOQ are not required for an ID assay. If a cIEF method is used for purity determination, then all the purity parameters shown in Section 4 should be qualified. The following sections illustrate an example of method development and qualification procedures for cIEF. 

The method of choice is dependent upon the analyte, the assay performance required to meet the intended application, the timeline, and cost-effectiveness. The assay requirements include sensitivity, selectivity, linearity, accuracy, precision, and method robustness. Assay sensitivity in general is in the order of IA > LC-MS/MS > HPLC, while selectivity is IA LC-MS/MS > HPLC. However, IA is an indirect method which measures the binding action instead of relying directly on the physico-chemical properties of the analyte. The IA response versus concentration curve follows a curvilinear relationship, and the results are inherently less precise than for the other two methods with linear concentration-response relationships. The method development time for IA is usually longer than that for LC/MS-MS, mainly because of the time required for the production and characterization of unique antibody reagents. Combinatorial tests to optimize multiple factors in several steps of some IA formats are more complicated, and also result in a longer method refinement time. The nature of IAs versus that of LC-MS/MS methods are compared in Table 6.1. However, once established, IA methods are sensitive, consistent, and very cost-effective for the analysis of large volumes of samples. The more expensive FTMS or TOF-MS methods can be used to complement IA on selectivity confirmation. 

Method robustness was established to show assay consistency with various supplies of the reference standards and two other critical reagents ... 

Robustness and ruggedness experiments are used to demonstrate how reproducible a method is when conditions vary. An assay method protocol defines the exact steps to be followed to ensure an accurate and precise result. Unfortunately, slight-to-large deviations occur in the everyday process, which may or may not impact the result. Assay conditions like incubation temperature or exposure to light define the robustness of the assay, whereas changes to the routine, for instance, multiple analysts or different instruments, define the ruggedness. The actual batch size for a routine sample analysis run is frequently overlooked but is often an impactful ruggedness measurement that should be assessed. 

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... 

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. 

During method development (Chapter 9) and validation (Chapter 10), QCs are used for several purposes including checks on precision and accuracy, lower limit of quantitation (LLOQ), recovery and method robustness and ruggedness (Section 9.8.4), as well as stability studies of various kinds (Sections 10.2.7 and 10.2.8), studies of inter-day validation within a specified laboratory and cross-validations in inter-laboratory method transfer (Section 10.2.11). QC samples are also used during method development to assess the final method prior to validation experimental runs that use QCs for this purpose are often referred to as assay prequalifications or pre-study assay evaluations (PSAE). 

Fluorescence-based methods do not directly measure ionic current but, rather, measure either membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals (from fluorescent dyes loaded into the cytosol or cell membrane) as a result of ionic flux. Because fluorescence-based methods give robust and homogeneous cell population measurement, these assays are relatively easy to set up and achieve high throughput. 

A recent study published by Badema et al. in 2011 describes a combined method to investigate the toxicity of an industrial landfill s leachate which is based on a triad approach including chemical analyses, risk assessment, and in vitro assays [17]. Moreover, to verify the applicability and the robustness of the proposed method, the approach was applied on a real case study a controlled, ISO-14001 certified landfill for nonhazardous industrial waste and residual waste from the treatment of MSW in northern Italy for which data on the presence of leachate contaminants are available from the last 11 years. 

The FRAP method is a simple, inexpensive, and robust spectrophotometric technique. However, the relevance of this assay is uncertain, because the assay reaction occurs by electron transfer, which does not mimic physiological situations. 

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