Selectivity bioanalytical assay

Given the foregoing discussion of some of the unique characteristics of macromolecules that lead to clear differences in their pharmacokinetics compared to those typical of small-molecule drugs, there is a subset of the entire group of bioanalytical assay validation parameters that are of key importance in support of pharmacokinetics of candidate macromolecular therapeutics. Assuming demonstration of accuracy and precision of sufficient quality for the intended application of the assay (e.g., non-GLP discovery support or GLP toxicokinetic support, as discussed above), the most important characteristics of a given assay in support of pharmacokinetic studies are likely to be selectivity, specificity, and reproducibility for analysis of incurred samples. These are all related to the ability of the LBA to detect and quantitate solely, or as closely as possible to solely, the analyte of interest. 

S.3 Selection of Metabolite(s) for Monitoring in Clinical Studies and Nonclinical Toxicological Studies The objective of the metabolite measurement using a bioanalytical assay, such as an LC/MS method, is to determine its pharmacokinetic parameters (such as AUC and Cmax values) of a specific metabolite. The MIST document recommends quantifying certain human plasma metabolites, including major metabolites, important active metabolites, and metabolites associated with structural alerts using a... 

Th for the precursor ion indicates interferences sufficiently serious that it is not clear where the analyte peak boundaries should be set in order to measure peak area in contrast, use of a peak width of 0.2 Th gave a much cleaner, well defined peak (Figure 6.12). This example is taken from an extensive examination (Jemal 2003a) of the performance of this enhanced resolution quadrupole system in the context of high throughput bioanalytical assays. While the increased selectivity was clearly established, it was also found that the enhanced resolution mode of operation requires considerably more attention... 

In any event, a strategy for demonstrating selectivity should be developed and included as a part of every method validation and the results should be presented and discussed in the final validation report (Section 10.3.3). The amount of tolerable interference will depend on the analytical objectives and on the needs of the end user of the data. For a bioanalytical assay, background interference of less than 20 % of the response at the LLOQ is often considered to be acceptable this criterion is related to the recommendation (Viswanathan 2007) that the analyte response at the LLOQ should be at least five times the response due to blank matrix. Laboratory SOPs will dictate how this is measured in practice. If just a single measurement was made for each, the ratio of area (or height) of the LLOQ vs that of the blank is used, but if rephcate measurements were made for the cahbration curve at each concentration (as in bioanalytical practice), the lowest of the responses at the LLOQ would most likely be used for this purpose. 

Due to its inherent selectivity and sensitivity, Uquid chromatography-tandem mass spectrometry (LC-MS/ MS) has become a tool of choice for quantitative bioanalytical assays that also prove to be fast and accurate [1,2], The first part of this chapter provides the current best industry practice of developing and vahdating an LC-MS/MS method and then applying it for sample... 

Today, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are considered the standard ionization techniques for LC-MS/MS due to their predominant advantages in quantitative analysis of drug molecules in various sample matrices with high sensitivity, selectivity, reliability, robustness, and ease of operation. Other techniques, for example, atmospheric pressure photoionization (APPI), electron capture atmospheric pressure chemical ionization (EC-APCI), and high-field asymmetric waveform ion mobility mass spectrometry (FAIMS) serve as complements to the established ESI and/or APCI technical platforms whenever necessary for an enhanced sensitivity and/or selectivity of a bioanalytical assay [4,5]. 

This chapter concerns arrays of electrodes, or electrochemical cells, that have been applied to bioanalytical assays. These arrays are orderly arrangements of elements, with a minimum of two, that are uniquely and individually addressable. Ensemble electrodes, in which all of the exposed macro-, micro-, or nano-sized sensing elements are connected to a single source of stimulus and measurement of response, are not considered, nor are electrode arrays that are used for electrical stimulation (for example, arrays used for cochlear implant stimulation) without measurement of an analyte-selective response. 

Close cooperation for a year or more before the first administration to humans is likely to lead to a smooth transfer of the compound and the rapid movement of a compoimd out of preclinical into man. This lead time can be used to devise the ED plan, design the first studies and, when appropriate, to select and develop methodologies that will contribute to the drug s evaluation in man. This may include validation of pharmacod)mamic measures to be used in the clinical pharmacology unit, assessment of various imaging techniques, development of bioanalytical methods. Not infrequently, the assays that were perfectly adequate to support preclinical work are insufficiently sensitive, specific or accurate to quantify the comparatively low concentrations of parent... 

Low permeability can itself be the cause of apparent discrepancies between biochemical and cell-based assays and may or may not have physiological relevance. Independent of the solubility limitation mentioned above, the selection of an appropriate loading concentration in cell-based permeability assays impacts on the assay outcome and depends on what information one wants to extract from the measurement loading at high concentration (i.e., 100 pM) will essentially cancel the effect of active transports unless passive diffusion is low. When high loading concentrations are used, poor recovery and bioanalytics are usually not an issue. 

The fundamental parameters for bioanalytical validations include accuracy, precision, selectivity, sensitivity, reproducibility, stability of the drug in the matrix under study storage conditions, range, recovery, and response function (see Section 8.2.1). These parameters are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation, such as selectivity and quantification issues. 

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