Incurred sample reanalysis: different evaluation approaches on data obtained for spironolactone and its active...

The inherent reproducibility of a bioanalytical approach is usually sustained through incurred sample reanalysis (ISR). Questions relating to the number of ISRs, the right moment for performing reanalysis, the way of performing an appropriate statistical refinement of experimental data and actions to be taken in the case of failure are frequently raised.

Data resulting from ISR following a bioequivalence study for spironolactone formulations are discussed. Reanalysis of samples was carried out twice: immediately after the end of the study and after a period that overcame the long-term stability study achieved during method validation. The Bland-Altman approach was used to assess experimental results. ISR was successful over the short reanalysis period for both compounds. Data produced through reanalysis after the long-term period indicated a systematic positive bias for the metabolite canrenone (although results supported reproducibility). The results obtained for spironolactone were affected by a strong negative systematic bias and failed to support reproducibility. The explanation deals with the continuous conversion of spironolactone to canrenone in plasma samples. However, reproducibility of the method may be sustained by comparing original and repeated differences between concentration values in samples by means of a paired t-test, Wilcoxon sign rank-sum test and linear regression.

Different statistical approaches for making data comparisons are discussed and may be successfully applied during reanalysis of samples from a bioequivalence study. Results of the evaluations may differ in accordance with the statistical procedure being applied, thus a definitive conclusion requires consideration of all specific experimental circumstances arising during production of the processed data.