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Standard addition calibration for small molecule

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Standard addition calibration for small molecule J Larose  2019-09-09 12:36
 

Hi,

I analyse several small molecules that are endogenous. I use the standard addition method to determine what is the initial concentration in the sample in which I spike my standard solutions to build my calibration curve. Then, I adjust the concentration of my curve points to take into account the actual concentration of the curve points, ie the endogenous concentration plus the added concentration. Is there a way to write this in the document grid? My compounds have different added concentrations so I use the concentration multiplier to correct that but I see nowhere where I can add a fix amount of each molecule to the concentration.

Thank you!
 
 
Nick Shulman responded:  2019-09-09 14:12
I do not understand your question.

It might be helpful if 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.
Otherwise, you can upload it here:
https://skyline.ms/files.url

Can you maybe show us a screenshot of what you are asking about?
-- Nick
 
J Larose responded:  2019-09-10 09:34
I put a picture of what I'm talking about. First, I use a technique called the standard addition method to determine what is the concentration of the endogenous compound in the matrix I use to build my calibration curve. Then, I sum this endogenous concentration to the one I add in the matrix. I finally use the real concentration (endogenous + added) to plot the calibration curve. I measure about 40 compounds at a time in my method and their concentration is different in each standard because of the endogenous part. I have a Sciex instrument and Analyst is too slow for quantification.

To summarize, it would be easier if it was possible to write separately the concentration of each compound in each standard. I know it is possible to do it by using the analyte concentration * concentration multiplier, but it seems that it is not possible to do an addition of a fix concentration.

I hope this is clearer :)

Also, is it possible to use the height ratio instead of the area ratio for the quantification?

Thank you!
 
Nick Shulman responded:  2019-09-11 13:08

Thank you for the diagram.
I see that you have these concentrations:

Replicate Compound A Conc Compound B Conc
STD0 5 ng/mL 8 ng/mL
STD20 25 ng/mL 48 ng/mL
STD40 45 ng/mL 88 ng/mL
STD60 65 ng/mL 128 ng/mL
STD80 85 ng/mL 168 ng/mL
STD100 105 ng/mL 208 ng/mL

In this case, there is no simple ratio between the concentration of compound A to compound B across the replicates.

Skyline does not currently support this, so you would have to deal with compound A and compound B in separate Skyline documents, or you would have to lie to Skyline about what the actual concentrations were and then add or subtract an appropriate number from the final result.

We have anticipated for a long time that Skyline might need to allow users to specify the "full matrix" of concentrations of compounds and replicates instead of just relying on the Replicate's "Analyte Concentration" and the Molecule's "Concentration Multiplier".

Are you planning on using the calibration curve in Skyline to calculate the concentration of unknown samples? If so, you should definitely send us your Skyline document. The main reason that Skyline does not have this "full matrix" feature yet is that we have not seen a dataset that required it, and so I was not confident that I could implement this and get the correct numbers without a datset to test it on.

Usually when scientists are requesting this "full matrix" feature, they just want to determine the linearity of the response curve. When it actually comes to using a calibration curve to quantify unknown samples, they always have been intending to use a single point calibrator.

Either way, it would be great to see your data.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.

If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:
https://skyline.ms/files.url
-- Nick