LOD calculation using internal standard

LOD calculation using internal standard ssaleh  2018-10-18


we spiked a sample matrix with a mixture of internal standard ( 58 labelled peptides) with different range of concentration. We then run the samples in triplicate on TSQ. The idea is to calculate the LOD.
I was wondering what should I use to build the calibration curve? Total area in precursor results (attached figure)? is there a way to do this in skyline or we should export the values and make the curve in Excel?
Could you please refer me to a tutorial in skyline where you did LOD calculation using IS.

Would like to share the skyline document if needed but in private mode.
hope you can help!


Nick Shulman responded:  2018-10-18
Skyline has the ability to make calibration curves. There are some tutorials about that here:

In Skyline-Daily, we have also added some features where you could calculate the LOD and LOQ based on things such as the standard deviation of your blanks.

If you are just interesting in exporting numbers to Excel to make your own calibration curve there, you probably want to use the column called "Ratio To Standard".
-- Nick
ssaleh responded:  2018-10-24
Dear Nick,

many thanks for your answer.
In the tutorial absolute quantification they indeed used Ratio to Standard parameter to make the calibration curve. However in my case I spiked the matrix with only heavy peptides. The idea is to get the LOD from the matrix and compare it to values of light in my sample of interest (so wed don't care about the amount of light in the matrix). In this case the ratio to standard parameter is not relevant, I guess I can use peak area Vs concentration to plot the curve and determine LOD?

But the Problem is when exporting the peak areas of my sample of interest and use it to calculate the concentration of the light using the equation (y= ax + b with y peak area, a the slope and b the intercept) I get unreasonable values.. can we compare peak areas in different TSQ runs?

one last question, how can I use skyline daily to calculate the LOD based on SD of the blanks?

many thanks in advance,
Nick Shulman responded:  2018-10-24
I do not know why the slope you calculated with your heavy spike in calibration curve would not produce meaningful numbers with your light data.

You should send us your Skyline documents and we will figure out what is going.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If those .zip files are less than 50MB then you can attach them to this support request.
Otherwise, you can upload them here:

Skyline-Daily added some new features related to calculating LOD and LOQ. To use this feature go to:
Settings > Peptide Settings > Quantification
at the bottom of the Quantification tab on that dialog, there is a "Figures of Merit" section where you can specify the maximum CV etc.
-- Nick