Orthogonal assays

Amplified Luminescence Proximity Homogeneous Assay (AlphaS-creen ) technology have been reported [50,51]. With an HTS methodology in hand and orthogonal assays previously developed [47-49], nonpeptide antagonists of methyl lysine binding should appear from diversity screening approaches in the near future. 

The accuracy of a method is defined as the closeness of the value obtained to known or accepted values. Accuracy can be determined in a number of ways, depending on the nature of the CZE method and availability of orthogonal techniques to compare results. If practical, spike recovery studies (i.e., testing to determine whether recovery matches the amount of a known analyte or impurity spiked) are good alternatives to orthogonal assay comparisons. ICH guidelines also allow method accuracy to be inferred, once specificity, linearity, and precision are established. 

Analytical chemists generally run a series of different assays (orthogonal assays) to confirm that they have sufficiently characterized the sample and have identified all impurity peaks found in the sample. Furthermore, they ensure that peaks which may co-elute using one technique can be detected with an orthogonal method (see Chapter 10). Analytical HPLC is the most common technology employed for assay and impurity profiling of pharmaceuticals. When the sample has been analyzed, the impurity may need to be isolated (for structural characterization, for instance). While techniques such as LC-MS may give an indication of the compound identification from the bulk assay, the definitive proof is always obtained from an independent analysis of the isolated compound. 

CRC, Counterscreen, Orthogonal Assay, Secondary Assay IC50... 

Fig. 32.5 - Profiling). Concurrently, if feasible, the hits or hit classes are submitted to orthogonal assays like electrophysiology, in the case of neuronal targets, that help to further classify and validate the hits independently of the readout. 

False positive technology hitters should be removed using an orthogonal assay. 

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