amount of internal standard working solution?

[noobie]

Hello,

I am struggling with a question which I have not been able to find an answer for a quite a while now. It is related to the amount of the internal standard working solution (not the concentration of it) you add to your calibration standards, QCs and study samples (plasma, urine) etc. prior sample extraction process.

I am working in a lab that is in charge of developing and validating methods for clinical PK analyses with LC-MS. The molecules we receive are the ones in development process, so I cannot rely on literature. I have to start from scratch on my own, with systematic method development process etc. And recently the question of the volume internal standard working solution with LLE and SPE extraction methods puzzled me.

This issue is not a problem for protein precipitation since the IS working solution is diluted in the prepitating organic solvent inducing precipitation but problem seems to arise with LLE and SPE. I have been looking some couple of articles from diverse publishers as well as some textbooks on LC-MS and bioanalytical validation but none of them gave me satisfactory answers. Some of them injected the IS working solution at the ratio of 1:1 (exm: 200 uL of calibration standard added with 200 uL of IS working solution) but in some others this ratio shifts to 4:1, 5:1 in favor of the samples (exm: 100 uL calibration standard with 25 uL of IS working solution).

How do you determine the volume (the amount) of the IS working solution for LLE and SPE when developing a method? Is there any general rule or some kind of "rule of thumb" to help in making the right decision concerning this issue? Of course one would say it depends on the solubility of the IS, but apart from that, is there anything that influences the volume of the IS working solution to choose?

Thank you for any help you can provide.

[Don_Hilton]

Volume selection includes a couple of considerations:

1) large enough to be able to alequot accurately
2) small enough to not overly dilute the sample and to be convienient for handling.

If the solvent for the working solution plays a role in the sample prep - such as buffering the sample, dilution prior to application to SPE, or such, then the volume depends on any intended chemistry with the sample.

[noobie]

Volume selection includes a couple of considerations:

1) large enough to be able to alequot accurately
2) small enough to not overly dilute the sample and to be convienient for handling.

If the solvent for the working solution plays a role in the sample prep - such as buffering the sample, dilution prior to application to SPE, or such, then the volume depends on any intended chemistry with the sample.

Thank you Don_Hilton for your reply.

So I guess it's "case-by case"...? It depends on each lab's SOP etc...?