Bioequivalence and Bioavailability Forum

Dear Jaime_R

Responding to your post.

❝ 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):

Variability does not occur in volume transfer. A good chemist can transfer the needed volume exactly.
It is the method of extraction that can or cannot guarantee to transfer the needed amount each time...!!!!!
detector response has "SOME THING" (1) External standard (ES) method to variability
1- What if I cant have a Standard similar to the analyte in chemical behavior?
-then I might apply or compromise my detection method for the favor of IS, ES
-I might need a lamda switching. ... variability is there.
2- What if my standard has low detection response?
-then I need to add a huge amount to the sample. ... variability is there.
-the matrix would be affected, and the lower concentrations will be affected in response. ... variability is there.

❝ 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.

Where exactly you add the ES...?

❝ (2) ES with problems

❝ e.g., a gel was formed during mixing, only 150ul 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.

If the method where developed well, then this e.g., would be an issue back then, nevertheless errors occur, and when they do, I just need to repeat this sample.

❝ (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).

But the detection response would not be consistent, thus reading the area or height for the lower points would be difficult and inaccurate...!!!

❝ The IS method is more convenient, because we don't have to bother about exact volume transfers.

❝ 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.

Well,, do you do full and partial validation to the same method for the same study???
"interfering peak" is there weather you applied ES or IS, how would the ES eliminates that "peak"...!!!!!

Regards
Charl