Bioequivalence and Bioavailability Forum

Dear Charl,

only responsing to part of you post.

❝ 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)...!!!

As far a I know - and stated by HS - the IS protects against variability in volume transfer. It has nothing to do with detector response.
Just to give an example (liquid/liquid extraction):
(1) External standard (ES) method
Exactly 500µl plasma + exactly 500µl organic → vortex → centrifuge → exactly 400µl organic to another vial → evaporate → dissolve in exactly 200µl mobile phase → autosampler: calculated amount depends on error of all volumes.
(2) ES with problems
e.g., a gel was formed during mixing, only 150µl organic can be transfered: the volume has to be measured - which is not that easy - and must be incorporated in calculation: calculated amount depends not only on the variability of volumes as in (1), but heavily on measurement error of the volume of organic.
(3) Internal standard (IS) method
As (1), but both transfers of organic don't have to be an exact volume, since the ratio of analyte/IS is employed in calculation - which remains constant with varying volumes.
(4) IS with problems
As (3), nothing has to be done in terms of calculation, although - as stated by Ohlbe - problems exist at low concentrations (but this is also true for all methods).

The IS method is more convenient, because we don't have to bother about exact volume transfers.
But: there are examples in the literature (please don't ask me where - buried somewhere in my files...), where the IS performed worse.

Routinely we do the following: During validation we perform all steps of an IS method as an ES method; in the validation report we include the entire data set in two 'flavours':
(1) the IS method as standard, and
(2) the ES method as an 'alternate'.
If (2) also fulfills generally accepted requirements, the method is validated for both.
In the analytical protocol we define a primary and an alternate method. If something goes wrong in routine analyses (i.e., a peak in a particular subject's samples interferring with the IS) we simply may switch to the ES method. Of course criteria have to be defined in an SOP or the protocol, and exact volume transfers have to be done.