Table 4. Based on the results of the specificity analysis (Table 3), we conclude that the specificity of the assay is 0.2
The use of standards, product, and spiked excipients for method validation depends on the purpose of the method under test.
If National Institute of Standards and Technology (NIST) or World Health Organization (WHO) standards exist for the test method,
or if a pharmacopeial method is under consideration, those standards should be used. Standards can be used if the test method
requires a standard curve to calculate the response. Bulk drug product or final drug product are used when stability indicating
assays are being tested. Stressed samples and time zero samples typically are used for method validation of these stability-indicating
assays. When novel proteins or unique products are being tested, typically the only samples that are available are spiked
samples. When using spiked samples, one must assume that the spiked value is known without error.
Table 5. In this example, the expected range for the assay was 50–150% of the nominal value. Results from using two analysts,
each testing five samples three times per day for two days.
The following example shows how specificity analysis would be conducted using the above protocol. Using four samples each
with six repeats, an equivocal zone of 0.375 to 0.465 would be used to determine if there are differences among the four levels
of analyte. For each level, one would compute the 95% confidence interval and compare it to the equivocal zone. Table 3 shows
the results. Based upon those results, we conclude that the specificity of the assay is 0.2 mg/dL (Table 4).
Table 6. A typical accuracy analysis
The validation of linearity, accuracy precision, and range was performed on a cell-based bioassay that did not require a level
of quantification or level of detection. The expected range for the assay was 50–150% of the nominal value. Using two analysts,
each testing five samples three times per day for two days yielded the results in Table 5.