Bioequivalence and Bioavailability Forum

Dear Harish, Ohlbe, & HS..

We should all go back to the concept of WHY do we need the IS.....!

as far as I know it is used to minimize the calculation errors raised by doing the Absolute peak (height or Area)...!!!

so... variability of IS should be at the minimum, it should be studied throughly while developening your method.
(e.g... as Ohlbe mentioned about LLOQ, the case will be due to only the area of the analyte (drug) that would vary to the 1/4, so having a minimal IS variability in this case, we will know that some thing went wrong).

from my personal experience.... I always think fresh, from scratch, for studying the variability of IS while developing, thus for each HPLC detector there is different approach, e.g, the electrochemical detector would be more sensitive than the UV, the LC MS/MS would take a different approach taking into account the ion enhancing and suppresing, matrix effect, etc.....

So finally I recommend that the IS variability is crucial, so we should do the simplest procedure of extraction, cause there were the most error would occur without noticing

regards....
Charl