Bioequivalence and Bioavailability Forum

❝ We suspected additional metabolizing steps due to freeze and thaw. This was not observed during method validation.

• if no sample was re-analysed during the study, or if samples were re-analysed using the second aliquot (whatever the reason for reanalysis: dilution, failed run, etc.), I would also use the second aliquot for incurred sample reanalysis, even if freeze/thaw stability was shown during method validation for 3 or 5 cycles. Reason: if you find a mean 12 % decrease after your F/T cycles you will still conclude that stability is OK - but a 12 % decrease, if real, could seriously increase your chances of failing the incurred sample reanalysis.
  
• if samples were re-analysed during the study using the first aliquot (which would not be my preferred option - and what would be the point in having two aliquots then ?), I would also do the incurred sample reanalysis using the first aliquot. If you have a problem with metabolite back-conversion during F/T cycles (which you will not see during the validation unless you also spike the samples with the metabolite), you will have more chances to see it. And that's precisely what regulators ask you to do the incurred sample reanalysis for.