Bioequivalence and Bioavailability Forum

Dear Harish!

❝ How much of Internal Standard (ISTD) Variation is allowed for bioanalysis by HPLC and LC-MS/MS?

During validation: anything that leads to a validated method.

Once you have set your specifications of the method, there’s no point looking at ‘Internal Standard Variation’. Plainly speaking: either a particular batch is valid or not.
The more similar an internal standard is in its physicochemical properties to the analyte, the less you should be concerned about varying detector responses.

Example: Sometimes cartridges get blocked in solid phase extraction (SPE), and only a small fraction of the usual volume will be obtained. If you are using a stable isotope IS in MS it simple wouldn’t make any difference. Of course your signal would be much lower, but who cares. On the other hand, if you are using a compound which is only fairly similar to the analyte, following situation may occur:
Although the elution from SPE-cartridges is expected to happen in an on/off manner (Agilent Technologies, formerly Hewlett-Packard Analytics called it ‘Digital Chromatography’ in the last century), in a partly blocked system the situation may be different. In the worst case all of the analyte or IS is still in the cartridge, whereas the respective other compound is already eluted. This is a pretty nasty situation. Therefore:

• choose an IS as similar as possible to the analyte.
  
• if blocking occurs, document it in the lab notebook.

But remember, if you get a ‘strange result’ based on the IS response, there's no way to resolve the problem from the chromatogram itself. The only remedy would be to run the entire method with constant volume transfers. In such a case it would be possible to track the error down to its source. But if doing so, one could also omit the IS at all, and go with an external standard only (no guideline forces us to use an IS!). See also this thread.

❝ Is there any guideline for this?

No, this term is not mentioned at all (there’s not a guideline for everything).

There is not difference in validation or routine analysis between HPLC and LC/MS (besides a higher likeliness of matrix effects in LC/MS). Guidelines treat only bioassays differently.