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Topic: Quantification relative vs quantification absolue (Read 401 times) previous topic - next topic

Quantification relative vs quantification absolue

Dear all,

Sorry for this basic question, but i need our advice about relative lipids quantifiation between two sample.
I'm beginner.

Imagine that we started with the same starting quantity of a sample (weight (ie 10 mg DW).
We wish to compare samples A versus B.
No internal standard during extraction
Injection using LC-QTOF ddmS2, data procesing using MS_Dial and Lipid blast for annotation.
Is it possible to perform a relative quantification between A and B (normalization to total amount of a lipid class or all measured lipid classes) ? or an internal standard need to be put during extraction process ?

Sorry for this basic question

Re: Quantification relative vs quantification absolue

Reply #1
Hi Sebas,

No problem is asking basic questions. That's why we are here!

So, ideally you would have had internal standards added from as early a step as possible. Given that you do not have that, it is possible to perform a 'relative quantitation', but there are a lot of caveats. Using internal standards allows a lot more than just quantification.

First thing, it's important that you have performed all the other steps appropriately:
  • The extraction was performed to minimize sample losses or changes in concentration. This is to ensure the signal you measure is as close to proportional to the original concentration as possible (all else being equal).
  • Ensure lipid loading is consistent. 10 mg dry weight can contain very different amounts of lipids depending on the sample type i.e. adipose tissue vs lean muscle. Normalizing to lipid classes isn't going to be accurate unless you are doing an 'apples to apples' comparison.
  • The samples were run in a randomized order, with appropriate quality controls run. If all of samples A were run before samples B, it's possible the intensity differences were due to technical issues, not biological.
  • If you run 'technical' replicates, ensure they are performed independently from the dry matter. Triplicate injections tell you about HPLC/MS variability; Triplicate extractions capture the whole process.

So interesting things to think about. If you did have internal standards and you measured 20 lipids in a class. Using the standard way of calculating concentration=[ISTD conc]*[peak area]/[ISTD area], for all measurements. Then, if you did normalize to the lipid class, you would get the same answer as if you completely ignored the internal standard!
Whenever you ratio concentrations that used the same internal standard, you remove the effect of the internal standard.

I hope this helps.

 

Re: Quantification relative vs quantification absolue

Reply #2
Hi CoreyG,

Thank you very much for this reply.