Selectivity

The selectivity (also referred to as specificity) is the ability of the assay to measure the drug or analyte without interference from other constituents in the sample matrix. In chromatographic systems, selectivity is demonstrated by comparing the detector response in the presence of drug, to a blank sample of plasma that was not exposed to the analyte (see Fig. 6). Comparisons of the chromatograms, and the peak area or heights between the drug and the blanks are made to demonstrate selectivity. Blank chromatograms should be obtained from sample matrix (e.g., plasma) obtained from six different sources that have not been treated with the drug. Furthermore, it is also advisable to determine whether any medications to be co-administered during the clinical study will interfere with the quantification of the analyte of interest. In addition, if an internal standard is used in the method, blanks with internal standard should also be compared to the drug and completely blank matrix to demonstrate that the internal standard will not interfere with analyte quantification. For other nonchromatographic types of analytical methods, such as RIAs and

FIGURE 6 Selectivity. The upper curve is a HPLC chromatogram of blank plasma. In the middle tracing, drug X and an internal standard (ISTD) were spiked into plasma. In comparison with the blank plasma, it can be concluded that the assay provides good selectivity for this drug. The bottom chromatogram is an example of assay in which the peak of interest (retention time of 10 min) is interfered with by a larger unknown peak.

FIGURE 6 Selectivity. The upper curve is a HPLC chromatogram of blank plasma. In the middle tracing, drug X and an internal standard (ISTD) were spiked into plasma. In comparison with the blank plasma, it can be concluded that the assay provides good selectivity for this drug. The bottom chromatogram is an example of assay in which the peak of interest (retention time of 10 min) is interfered with by a larger unknown peak.

ELISAs, the demonstration of selectivity is more difficult because there is no visual representation of the assay. In ligand-binding assays, an antibody binds to some chemical entity, and quantification is based on some radioactive tracer or enzyme activity. However, how does one know that the antibody does not bind some entity other than the analyte of interest? In these cases, the best assessment of selectivity is made by screening ligand crossreactivity with other compounds known to be chemically similar to the drug (i.e., endogenous compounds, drug fragments, etc.). The difficulty is there may be interactions with compounds that are not predictable. Therefore, the selectivity cannot be known absolutely with these methods. In these cases, it also recommended that selectivity of the ligand-based assays should be confirmed with the use of other analytical methods that rely on different principles (e.g., HPLC). In addition, nonspecific binding of the ligand may occur, and the prozone effect, i.e., nonspecific binding with buffer constituents, should also be assessed regularly [11].

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