Calibration Curve Molecules Ratio to heavy

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Calibration Curve Molecules Ratio to heavy sophia_foehr  2023-06-01 07:34
 

Hello,
we are trying to make a calibration curve with 4 internal standards for 11 analytes. It works if we set one to “Global Standard” and use “Ratio to Global Standard”, but if we use “Surrogate Standard” and “Ratio to Heavy” we get “Error: All of the external standards are missing one or more peaks.”. If we use “none” “Normalization method” we also get a calibration curve.
We tried with the internal standard as within the analyte and separated, neither works.
We are using Skyline-daily 64-bit version 22.2.1.501.
I attached the file and hope for a solution.
Thank you very much,
Regards,
Sophia

 
 
Nick Shulman responded:  2023-06-01 07:51
The reason that you are not getting a calibration curve for some of your molecules is that those molecules only have heavy precursors.
"Heavy" has been designated an "Internal Standard" at "Settings > Molecule Settings > Labels".
Skyline will not use peak areas from internal standards when plotting a calibration curve because it is assumed that the internal standard was spiked into each sample in the same amount.
If you would like to see calibration curves for your heavy molecules you should go to:
Settings > Molecule Settings > Labels
and uncheck the box next to "Heavy".

By the way, when you designate a molecule as a "Surrogate Standard", that does not in itself change the normalization method of any molecule in the document. When something has been designated a surrogate standard, what it does is make it so that a "Ratio to surrogate Xxx" option becomes available to choose in the "Normalization Method" column in the Document Grid.

There is a little bit of information about surrogate standards on this Tip page:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=Surrogate%20Standards

-- Nick
 
sophia_foehr responded:  2023-06-02 01:09
Good morning Nick,

Thank you very much, your information helped!!!

Now I was wondering whether it would be possible to have different analyte concentration for each of the acids? The thing is we are using different concentrations for our internal standard and if we look at the calibration curve, it looks funny/wrong for some due to the different values. Is there a way to do that?

Thanks again,

Sophia