I want to add something that might be relevant.

When I plot the absolute response of the analyte or internal standard, there is non-linearity in both plots. There is a supression of the analyte on the IS response and visa versa. At least, that is what I conclude from that given information. However, when I plot the ratio, the non-linearity starts at a higher concentration. Which make me believe that the ratio corrects for suppression/saturation to a certain degree.

Mr. Asali,

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Use of an internal standard (IS) requires the response to that compoumd to be linear - this is the assumption when taking analyte/IS ratios. If you take a fixed ratio of analyte to IS and inject different amounts then that ratio MUST remain constant otherwise the the use of IS is pointless. If there is ion suppresion, or enhancement, and the ratio changes then the IS isn't compensating for the suppression and your results will be in error. Do your calculations with and without IS.

Bojidarka B. Ivanova Thank you for your answer. However, I am searching for a mechanistic explanation for the improve of linearity observed when increasing the internal standard concentration.

Robin Whelpton thank you for your answer as well. Im not sure if I have understood your answer correclty, but as I mentioned in the comment, when looking at absolute responses you see nonlinearity starting at lower concentration than when looking at the ratio between analyte/co-eluting IS. Which is expected by definition with the use of such IS. However, nonlinearity is not uncommon by increasing analyte concentrations. In literature, this is explained by either saturation of excess charge on the droplet surface in ESI, limited space on the droplet surface, or cluster/multimer formation of the analyte with itself and/or other species including the IS. The main question remains: how does increasing the internal standard improve linearity when taking into account the mechanisms of saturation with increasing analyte concentrations?

It may be that, since ESI is a competitive process, a high concentration of a co-eluting IS actually suppresses the formation of analyte's dimer/trimer in the ion source...you may check this by comapring the dimer/trimer signal (detected with their respective MRM) in both conditions (low conc vs high conc IS) for the whole set of calibrants. I would even expect that the use of too high conc IS would lead to a higher (worse) LOD for the analyte...is it?

Hello

Is there any guidelines concentration of IS added to the standard?

A rule of thumb is enough to give about a signal that is 50% of the signal given by the highest calibration standard - half way up the calibration curve as it were.