Bioequivalence and Bioavailability Forum

Dear Charl!

❝ Variability does not occur in volume transfer. A good chemist can transfer the needed volume exactly.

If your chemist is a human being, he/she will not be protected against errors, being a good one or not. If there's a small air bubble unoticed in the pipet it will not effect the result after addition of the IS, whereas in an ES-method it may lead to errors in all consequent steps.

❝ It is the method of extraction that can or cannot guarantee to transfer the needed amount each time...!!!!!

Yes, this favors SPE over liquid-liquid-extraction.

❝ 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.

Sure, but what are you trying to say?

❝ ❝ Exactly 500ul plasma + exactly 500ul organic -> vortex -> centrifuge -> exactly 400ul organic to another vial -> evaporate -> dissolve in exactly 200ul mobile phase -> autosampler: calculated amount depends on error of all volumes.

❝

❝ Where exactly you add the ES...?

Me personally? Depends on the method, but there's no difference whether an IS- or ES-method is applied.
For daily fresh prepared calibrators just spiking plasma, or diluting it (mainly for SPE) 1:x with buffered stock solutions to yield the target concentrations.

❝ ❝ (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.

❝

❝ 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.

Very nice. If a gel has formed, the reason probably (likely?) is a plasma constituent (lipid?) in that particular sample. Maybe re-analysis will solve your problem, but I'm having some doubts.
If you don't want to waste your back-ups, why don't you simply correct in numerically?

❝ ❝ 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...!!!

Yes, but so what?

❝ ❝ 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???

OK, this is definitely a misunderstanding (you used 3 question marks)...
We perform the full validation of all IS-methods as if they were ES-methods (i.e. taking care about exact volume transfers). By this we kill two birds with one stone:

• calculating calibration / QCs, whatsover by ratios analyte/IS: the method is validated for IS use
  
• calculating calibration /QCs etc by peak areas / heights (ignoring the IS): the method is validated for ES use

Nothing has to be done in terms of additional laboratory work; it's just a little bit sitting by the desk working on your PC.

❝ "interfering peak" is there weather you applied ES or IS, how would the ES eliminates that "peak"...!!!!!

Don't quote me wrong, I said:

❝ [...] (i.e., a peak in a particular subject's samples interferring with the IS)

I meant (and talked about) a peak interferring with the IS, not the analyte. If you are observing an interference at the retention time of the IS and your method is also validated as an ES-method and you have stated this in the protocol (better than in an SOP, because this is more transparent to inspectors) you may simply run this batch based on the ES-method. By this it's not even a protocol deviation; just report it.

If you are observing an interference with the analyte you will have to modify the method and do a partly re-validation (linearity, etc).

P.S. Please keep your quotes short (I'm not sure whether I answered your points correctly - so I kept them mainly in my post); single exclamation and question marks are suitable to get the emphases.