LoD higher than LoQ with small molecule analysis

LoD higher than LoQ with small molecule analysis ychiu2  2021-06-29

Hi skyline team,
I ran into an issue that for some reason the software calculate the LOD much higher than the LoQ. I am using labeled standard and choose "normalize to heavy" for the quantification and I have loaded three solvent blanks and denotes those as blank. I am not sure if it is because the labeled standard or some of the setting I have was wrong so that my Lod is higher than loq. Can I get a LoD in this condition?

My quantification setting is below
Regression fit: linear
Normalized method: ratio to heavy
Regression weighting: 1/x2
MS level: all

Max LOQ CV: 30%
Calculated LOD by: blank +- 3 SD
Qualitative ion ratio threshold: 30%

Thank you

Nick Shulman responded:  2021-06-29
If you are doing "ratio to heavy", you need to make sure that your "Blank" replicates do have the heavy standard spiked into them.

If a replicate did not have any heavy standard in it, the numbers that get plotted on the calibration curve would essentially be random numbers (noise divided by noise), and you would not get the results that you wanted.
If a sample contains neither heavy nor light material, it should be called "Double Blank", and it will not be used for the LOD calculation.

It is certainly possible to have a LOD which is higher than the LOQ.
The LOD and LOQ are calculated in very different ways, and Skyline does not do anything to prevent one number from being higher than the other.

If you still have questions, you can 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 an attach it to this support request. Otherwise you can upload it here:

-- Nick