Stable isopote dilution question

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AlbertJunges's picture
Stable isopote dilution question

Dear all,

I have a question regarding the isotope dilution method for correction of technical variability during extraction and measurements in LC-MS/MS experiments. I am constructing calibration curves based on (analyte area)/(deuterium labeled IS area) to determine the concentration of a given analyte in a given sample.

I am now not sure whether I should plot responses against initial concentration (that is the nM before adding the ISTD) or should I plot the final concentration take into account the dilution caused by additing the   ISTD mix? Does adding of ISTD is considered a dilution step or just a way to get the response ratio in a given concentration. 

Could you please also share some papers or references from where I can learn more about it?

Thanks a lot for your time.  I really appreciate it.



Dr. Analytical
Dr. Analytical's picture
Dear Albert:

Dear Albert:
If the entire sample that you spiked was processed through the procedure, then you should use the initial concentration, without dilution.  However, if these are just standards that you are injecting immediately after mixing, then use the diluted value.

Generally, we want to design a procedure so that this adjustment for dilution is not necessary.  If you use a more concentrated solution of IS (100Xanalyte), then you could add a similar amount without the need for adjustment (1% change is usually not important). 

Or, you could dilute the standards and IS solution to a constant volume.  For example, you could prepare three standards by adding 1 mL, 3 mL, and 9 mL to three 10 mL volumetric flasks.  To each flask add 1 mL of IS solution and dilute all to volume.  The concentration of IS will be identical in each solution, and the analyte will be present at the 0.1, 0.3, and 0.9X the stock solution.