Method Development, Validation, and Sample Analysis for Regulated Quantitative Bioanalysis Using LC-MS/MS - LC-MS in Drug Bioanalysis - page 63

Paper published in September, 2007 by Rocci et al. entitled “Confirmatory Reanalysis
of Incurred Bioanalytical Samples” [ 24 ] .
Based on the recommendation of Rocci et al.'s publication and other regulatory
documentations, the following section summarizes the main features which are
essential for ISR practice:

Type of studies . Incurred sample reanalysis is applicable to bioequivalence, phase
I-IV human trials and GLP preclinical studies, or any study with a unique popula-
tion. For GLP preclinical studies, the ISR evaluation only needs to be performed
once per method, per species.

Number of ISR samples . A minimum of 20 ISR samples for small studies (<200
study samples), at least 10 % ISR samples for medium sized studies (201-999 study
samples), with an additional 5 % ISR samples for larger studies (>1,000 study
samples).

Selection of ISR samples . ISR samples should be chosen from samples with report-
able concentrations at approximately >3 times the LLOQ, mid-range, and near the
approximate C max .
Dilution scheme . Whenever possible, a sample requiring dilution to yield a report-
able result should be reanalyzed using the same dilution factor as the original analy-
sis. Samples should not be diluted to levels <3× the LLOQ.

Timing of analysis . ISRs will be run within a time frame reasonably close to the
original analysis to allow real-time identification of potential problems and reduce
the impact that stability may have on reassay results.

Acceptance criteria . Two-thirds (67 %) of the ISR samples repeat values must be
within ±20 % of the mean of original and ISR values.

Reporting . A comparison table of results showing original result, ISR result and %
bias will be presented in the Sample Analysis report, along with the overall percent-
age of ISR samples meeting the acceptance criteria. A conclusion statement will be
included in the report text indicating if ISR met the acceptance criteria including
any follow-up actions if applicable.

Investigation . If ISR acceptance criteria are not met, an investigation is conducted.
Future analysis will not be conducted until management is confident that the assay
is capable of producing reliable and reproducible results.

8 Conclusion

Robust and rugged LC-MS/MS methods are essential in support of drug discovery,
toxicology studies, and clinical trials, for the data generated from these bioanalytical
methods is used to evaluate the bioavailability, bioequivalence, toxicokinetic, and
pharmacokinetic parameters of drug candidates. Thus, it is critical to invest significant
thought and effort in the method development process [ 25- 27 ] . Fast sample