How to caculate the Precision,Extraction recovery or Matrix effect with skyline?

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How to caculate the Precision,Extraction recovery or Matrix effect with skyline? kaylee xu  2020-02-11
 

Dear skyline team,

Q1: Recently, I am using Skyline for small molecular quantitative analysis. At methodological validation, I don't know how to calculated the Precision, Extraction recovery or Matrix effect with Skyline? Can you give me some steps?

Q2: If I build 10 compounds' calibrations at a time with different concentrations, eg. Compound-1( 1, 2, 5, 10, 20, 50, 100ng/mL),compound-2(2,4,10,20,40,100,200ng/mL) et.al. How to enter the concentration of these different compounds in the skyline?

Q3.What does these black error bar mean? Please see attach file.

Q4:Can I quantity one analyte in unknow samples with other analyte's standard curve line ? For example, I want quantity SM18:1 in unknown samples with SM18:1(d9)'s standard curve line.

Thank You!

Kaylee

 
 
Mike MacCoss responded:  2020-02-11

Hi Kaylee,
I will try and answer your questions one by one.

Q1: I'm not entirely sure how to answer your question. I don't think this question is really for Skyline. There are lots of reports that you can generate in Skyline. Using measured peak areas from replicate measurements you can calculate the precision as a standard deviation. The extraction recovery will require adding in a known amount of material and then assessing recovery of the spiked analyte. For matrix effects you would spike in common interferences and measure the concentration with and without the spiked interferent. None of these have anything to do with Skyline itself.

Q2: You might want to look at some of the tutorials and webinars. https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_small_quant
and https://skyline.ms/project/home/software/Skyline/events/2016 Webinars/Webinar 13/begin.view?
and https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/AbsoluteQuant-3_5.pdf

Q3: Width at half maximum.

Q4: It looks like you might be asking if you can use a stable isotope labeled internal standard to calibrate an unlabeled analyte. Yes you can use a stable isotope labeled internal standard as a single point calibrator.

-Mike

 
Nick Shulman responded:  2020-02-11
A couple of things to add to Mike's answer:

Q2: If one of your compounds was spiked in at twice the concentration as another compound, you would fill in the "Analyte Concentration" for each of the replicates with the smaller of the two concentrations (1, 2, 5, 10, 20, 50, 100)
Then, you would specify that the "Concentration Multiplier" of the more concentrated compound is "2".
The "Concentration Multiplier" column can be found in the Document Grid at:
Molecule Lists > Molecules > Concentration Multiplier

Q4: I do not believe that Skyline has any features to make it easy to use one molecule's calibration curve with some other molecule. This is a calculation that you would need to do yourself in some other tool such as Microsoft Excel.

By the way, if you would like to learn more about the Document Grid and exporting reports to other tools such as Excel you should take a look at the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

-- Nick