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Error: All of the external standards are missing one or more peaks

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Error: All of the external standards are missing one or more peaks szczepankaluzny  2025-03-12 09:03
 

Hello Skyline,

I hope you are doing well.

I would greatly appreciate assistance regarding the quantification of some compounds I am analyzing, please?

I am trying to quantify multiple analytes which were spiked in my standard with different concentrations.
I prepared the transition list and sorted the compounds with their internals standards using "Molecule List Name" adding "heavy" label type for my internal standards (see file attached).

Following that I adjusted Molecule Quantification Settings for Linear Regression with Ratio to Heavy normalization and regression weight of 1/x.

Then, I assigned the Sample Types and Analyte concentration to my raw data with Concertation Multiplier for my analytes of interest.

However all I am getting is "Error: All of the external standards are missing one or more peaks" when checking the calibration curve.
However I can see that the peaks are detected in my standards and unknown samples.

I would really appreciate some help,

Thank you,
S
 
 
szczepankaluzny responded:  2025-03-12 10:11

Hi there:
Just wanted to give an update

When I change the Normalization Method to "None", I am getting the standard curve based on the Peak Area. So I am wondering if that has something to do with how I assign internal standards.
Thanks
 
Nick Shulman responded:  2025-03-12 19:45
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.

Files which are less than 50MB can be attached to these support requests.
You can always upload larger files here:
https://skyline.ms/files.url

One problem that Skyline has is if you have not chosen a normalization method, Skyline assumes you just want to use the light transitions for the Calibration Curve. This is because Skyline assumes that the internal standard is going to be present in all of the samples at the same concentration so you wouldn't want to use them for a calibration curve. What Skyline means to say is "I can't figure out which transitions to say", but, instead, it says "All of the external standards are missing one or more peaks".
The way to fix this problem would be to go to the "Labels" tab at "Settings > Molecule Settings" and make sure to uncheck the checkbox next to the label type you want to plot in your calibration curve.
-- Nick
 
szczepankaluzny responded:  2025-03-13 03:47
Hello Nick,

Thank you for your response!

I followed your advice and did as you say regarding the label types selection in Molecules settings but unfortunately It did not change anything.

The problem I also noticed earlier is that I had standard "0" assigned as "Standard" even though it did not showed any peaks which might have been causing an error. Changing it to the BLANK helped.

However I am steal unable to obtain Peak Area ratio of light:havey. I have a feeling that the way I designed the transition list or Document Grid is potentially wrong.

I have shared Skyline document with you.

Best wishes
Stephen
 
Nick Shulman responded:  2025-03-13 08:21
In your transition list, the light and heavy molecules should have exactly the same "Molecule List Name" and "Molecule Name".
So, the Cortisol and Cortisone in your transition list might look like this:
Molecule List name,Molecule name,Label type,Precursor m/z,Precursor charge,Product m/z,Product charge,Cone Voltage,Explicit Collision Energy
1,Cortisol (F),,363.1,1,121.2,1,76,31
1,Cortisol (F),,363.1,1,91,1,76,83
1,Cortisol (F),heavy,366.17,1,124,1,76,31
2,Cortisone (E) 1,,361.1,1,163.1,1,81,31
2,Cortisone (E) 2,,361.1,1,77,1,81,107
2,Cortisone (E),heavy,364.17,1,166,1,81,31

Also, things work better if the light and heavy precursors have the same set of transitions. It looks like you are only monitoring two transitions for the light molecule and one transition for the heavy transition. You can make this work but it does involve checking the "Simple precursor ratios" like you have done on the Quantification tab at "Settings > Molecule Settings". Also, you should mark one of the light transitions as non-quantitative by right-clicking on it in the Targets tree and unchecking the "Quantitative" menu item.

When the "Regression weighting" on the "Quantification" tab at "Settings > Molecule Settings" is "1/x" you will get nonsense results if any of your external standards have a concentration which is 0.

-- Nick
 
szczepankaluzny responded:  2025-03-13 08:40
Thank you very much Nick for your time and all the help!

Let me try this out to see how it works!

Best wishes
Stephen