Precision. For early-phase methods, only injection and analysis repeatability is examined. Area % relative standard deviation (RSD)
is typically determined from 5 replicates. Repeatability is determined at 100% of nominal concentration for the API with impurities
being evaluated at the reporting threshold using the API as a surrogate. Acceptance criteria of 1% RSD (injection repeatability)
or 2% RSD (analysis repeatability) for API are frequently targeted. For impurities, higher precision limits (e.g., 10–20%)
are acceptable and should consider the level of the impurity being measured (injection and analysis repeatability). For tests
where the measurements are made at different concentrations (versus at a nominal concentration), such as dissolution testing,
it may be necessary to evaluate repeatability at more than one level.
Limit of detection and limit of quantitation. A sensitivity assessment is necessary to determine the level at which impurities can be observed. Using the API as a surrogate,
a "practical" assessment can be made by demonstrating that the signal of a sample prepared at the reporting threshold produces
a signal-to-noise ratio of greater than 10. A limit of quantitation can be determined from this assessment by calculating
the concentration that would be required to produce a signal to noise ratio of 10:1. Similarly, a limit of detection can be
calculated as the concentration that would produce a signal-to-noise ratio of 3:1. However, it is emphasized that the "practical
limit of quantitation" at which it is verified that the lowest level of interest (reporting threshold) provides a signal at
least 10 times noise and thus can be quantitated, is of paramount importance.
Linearity. Linearity can be determined from 3-point calibration curves at test concentrations of 70, 100, and 130% of nominal (API)
for assay or from 3 points ranging from the reporting threshold to 130% of the specification limit for impurities. API is
used as the surrogate analyte for impurities. For both analyses, a validation criterion can be established as R2 > 0.995. For tests where the measurements are needed over broader concentration ranges, such as dissolution testing, a broader
linear range may be examined using a 3-point calibration.
Range. As for late-phase methods, the range is inferred from the accuracy, precision, and linearity studies.
Robustness. Full robustness testing is not conducted during early development. However, an assessment of solution stability should be
conducted to demonstrate the viable lifetime of standards and samples. Specifically, solutions should be considered stable
when the following conditions are met:
- The API assay changes by not more than 2%
- No new impurities greater than the reporting threshold are observed
- Impurities at the reporting threshold change by not more than 30%; impurities at levels between the reporting threshold and
the specification limit change by not more than 20%; and impurities at or above the specification limit change by not more
Notably, if validation is performed concurrently with sample analysis as an extended system suitability, solution stability
must be assessed separately. This assessment is typically conducted as part of method development.