Hey All,
I am new to lipid biochemistry. I am planning to use odd chain fatty acid (OCFA) as internal standard for extraction (C17:0, C19:0 etc). Is it still relevant for quantitation. I have seen some of the articles showing relationship of OCFAs with diseases. I need you suggestions about using internal standards. Shall I go with OCFA or OCFA contatining lipids as internal standards?
Thank you for your response
There are actually two questions in one here: whether OCFA are OK as internal standard for FA quantification, and whether just free fatty acids are OK internal standard for quantification of FA from acylglycerides and phosphoglycerides.
For the first one, in my view, it will depend on the tissue/species you are working with. C15:0 and C17:0 are definitively present in detectable amounts in many species (specially ruminants, but not only). C13:0 could be a better candidate, but you should check first the literature about your target type of sample.
For the second one (whether the IS should be used as a free fatty acid form or as a tryglyceride or both), it might depend on some assumptions. First, as long as lipid extraction is concerned, by using a FFA as internal standard you are assuming that FFA, TG and PL are fully (or equally) extracted; and this is not true for most common lipid extraction methods (Folch and the like) even if they are adequately optimized, since usually, some PL families are not fully extracted.
And second, you are assuming that FFA, TG and PL are equally derivitized into methyl esters (or whatever derivative you are aiming for), and again, this is not true, since different methylation methods show different efficiencies for different lipid families.
Once said that, it will very much depends on how accurate you want to be. In animal science and food science, OCFA in their free form are very much used as extraction and chromatographic IS, because the small mistakes occasioned by these incorrect assumptions are supposed to be not relevant in the final outcome. However, in biochemistry or physiology, some of these small mistakes could be relevant.
Cheers
Dear Sangita,
In addition to Jorge's comments, OCFA are suitable lipid standards to quantify FA in most mammalian samples but not plants, viruses or bacteria. Although biosynthetic pathways synthesize even numbered carbon chains in mammalian, OCFA will also be present in human samples in result of diet intake. Based on this you should carefully choose your OCFA. In case you're aiming at quantifying FA in total lipid extracts you can release FA attached to tryglicerides and phospholipids by saponification reaction and then derivatize the hydrolyzed FA by transmethylation (FAME). Pls beware that not all FA will derivatize with the same efficiency. Hope this helps,
Ana
Any OCFA is very useful for plant lipid ... because OCFA is rare in most plant lipids..
but for animal lipid (mainly brain/liver ...) may use c13 not c17/c19..
OCFA contatining TG may be used (for plant/seed etc ..) if the sample is TG rich ..
responce factor, hydrophobicity of OCFA should be comparable for isolation of long chain fatty acids (neglect if it is minor ..)..
Dear All,
Thank you for your response.
First let me make clear about my question. I wanted to talk about odd chain fatty acid containing lipid (PC, PE etc). However I got answer on using fatty acid which is good. Thank you.
My concern is if diet contains odd chain fatty acid containing lipid, is it still good decision to use odd chain as internal standard for human sample (Ref to Ana's reply : OCFA will also be present in human samples in result of diet intake)? How can I discriminate between the odd chain lipid from diet (sample) or internal standard? For example if my internal standard is PC(17:0/17:0) and from diet if in my serum sample I get the same PC (17:0/17:0), is not that going to interfere with my quantitation?
The other question is " Can anyone share me the method or protocol to quantitate the lipid using mass spec? I want something like calibration curve or mathematical calculation protocol for lipid quantitation using mass spec.
I hope the experts here will help me to forward myself in lipid world.
Note: I am working with human samples like serum, plasma, tissues.
Thank you again for nice answers.
Best,
Sangita
Dear all,
i am also facing problem to quantitate fatty acid. is there any method or formula to calculate the amount of fatty acid?
I am new to use GC-FID and HLPC-UV, could you please someone can tell
what is the best method for both GC-FID and HLPC -UV for qualitative and quantitative analysis of FAME/biodiesel production obtained from transesterification of sunflower oil.
what type of internal standard will be using ?
how the sample will prepare for GC-FID and HLPC-UV?
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