Bioequivalence and Bioavailability Forum

Dear V.Pardhasaradhi!

❝ It is always a good practice to take plasma sample first into the vial and

❝ add internal standard to it.

True, because you easily notice whether you have missed a vial or not...

❝ ... Hence, it is better to practically prove (as you said, as part of

❝ your Robustness experiments) that it makes no significant difference

❝ whether you add 'IS to plasma' or 'plasma to IS'.

I strongly disagree with you.
IMHO (of analysing tenthousands of samples myself) missing IS addition is a rather rare event - I would guess <1/500... Any type of rubustness experiment does only make sense, if you get some meaningful insights of the influential parameters - in Roshini's case the order of IS addition.
From a statistical point of view this would mean setting up an experiment being able to detect a difference in the occurance rate of a rare event. Not going into the details of sample size estimation this would mean analysing a couple of thousand samples under both conditions. Of course this is not feasible. On the other hand, if you set up a kind of rubustness study in just a few samples (let's say the usual batch size for three days), the chance of finding a difference in conditions is practically nil. But since the power of such a study is small you can't claim anything from it:
Absence of evidence is not evidence of absence.

❝ ... better to practically prove ...

BTW, in science it's not possible to prove something. We only reject or accept a hypothesis.

Therefore I'm only with your second suggestion:

❝ ... you need to state in your method protocol or method SOP, what you

❝ planned to do....