Relative quantification with surrogate standards

Relative quantification with surrogate standards clay davis  2022-05-20

I am trying to quantify different lipid classes using several isotopically labeled surrogate standards. Once the labeled lipid internal standard is chosen as "surrogate standard" and set the "Normalization Method" to the respective standard for the other lipids, the normalized area reports n/a. I’ve looked at several ratio outputs in the report option but only the “Total Area Normalized” (Molecule list – Molecules – Precursors – Precursor Results and Area Normalize (Molecule list – Molecules – Precursors – Transition Results) seems to output a value as a percent and not necessarily the ratio or ratio x IS concentration.

  • Clay
Nick Shulman responded:  2022-05-20
Can you send us your Skyline document?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms. If that .zip file is less than 50MB you can attach it to this support request. You can upload larger files here:

I would expect that "Normalized Area" to have the numbers you want in it. I am not sure what might be going wrong but I will be able to figure it out after I see your document.

The "Area Normalized" column is not a useful column. It was added to Skyline a long time ago and is only available for backwards compatibility reasons and is calculated by dividing the transition area by the sum of the areas for all the precursors in the document which is very different from the number you want.
clay davis responded:  2022-05-20
Thanks for the quick response, I just uploaded the file (
Nick Shulman responded:  2022-05-20
Thank you for sending that file.
You should go to:
Settings > Molecule Settings > Labels
and uncheck the box next to "light" in the Internal Standards box.

The problem is that we make the assumption that anything which has been marked as an internal standard has been spiked into each replicate in the same amount, and so should not be used for quantification.
-- Nick