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Quantification of small molecules with internal standard

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Quantification of small molecules with internal standard viktoria thomanek  2026-03-09 03:22
 

Hi!
I am trying to quantify CoA-thioester after LC-MS analysis. The internal standard I am using is not a synthetic one of known concentration but a mixture of 13C CoAs extracted from cells grown on 13C substrate. What we do is, we add the heavy standard to our samples before sample preparation. Afterwards, we measure the heavy internal standard and define these areas as 100 %. When we measure the samples with the heavy standard, we get a relative ratio between the heavy standard before and after sample preparation and can determine how much sample is lost during preparation. This loss is added to our quantification results to calculate the "real" concentrations of our CoAs in the cell at the sampling time point.

My question now is, how can I quantify my samples in Skyline with this internal standard. My idea was to use a calibration curve to quantify the heavy standard and then in a second file quantify my samples with the known concentration of the heavy standard without a calibration curve. Is that possible or is there an easier option? Also, how can I quantify heavy peaks with a light calibration curve?

I added 3 Skyline Files. The first contains all samples, the 2. File is my attempt at quantifying the heavy standard and the 3. File is my attempt at quantifying my samples with the heavy standard. Can I trust the calculated concentrations in the document grid?

Thank you, for your help!
 
 
Nick Shulman responded:  2026-03-09 16:58
Your document "File 3_quantification of samples with heavy standard.zip" looks right.
On the "Quantification" tab at "Settings > Molecule Settings", the Regression Fit is "None" and the Normalization Method is "Ratio to Heavy".
Also, the "Internal Standard Concentration" has been specified for all molecules.
The "Calculated Concentration" will be the light:heavy ratio multiplied by the internal standard concentration.
This will give you fairly accurate concentration values, assuming the internal standard concentration value is correct. It relies on the assumption that the observed signal for the light molecule will be the same as for the heavy molecule. This assumption is usually a good assumption, but the numbers are always a little bit different because of things like isotope distribution and interference.

I do not understand what the samples were like in "File 2_quantification of heavy standard.zip".
When the normalization method is "None", the Y-axis values on the Calibration Curve will be the peak areas on the things which are not internal standards.
On the "Labels" tab at "Settings > Molecule Settings", there is a checkbox next to "heavy".
That means that the values you see plotted on the calibration curve will just be the light peak areas.
The reason that Skyline only plots the light peak areas is that Skyline assumes that the concentration of the internal standard will be the same in all replicates.
If you uncheck the checkbox next to "heavy" on the "Labels" tab, then the values in the calibration curve will be the sum of the light and heavy peak areas.

If you are trying to do a reverse calibration curve, then you should check the checkbox next to "light" and uncheck the checkbox next to "heavy". Then, on the "Quantification" tab you should choose "Ratio to light" as the Normalization Method.
However, you would only want to use "Ratio to light" as the Normalization Method if you the light concentration in each of the replicates were nominally the same.

I am not sure exactly what you are trying to do so I am not sure what your settings in this document should be.

You can find some advice about reverse calibration curves here:
https://skyline.ms/announcements/home/support/thread.view?rowId=36322
-- Nick
 
viktoria thomanek responded:  2026-03-16 07:39
Thanks for your help!

What I was trying to do in File 2, was to use the light standard to create a calibration curve and to quantify the haevy standard concentrations, because I do not know the exact concentrations of the heavy standards. What settings do I need for this?