Chromatography Forum

I am analysing a fatty acid derivative in plasma from preclinical species by ESI LC-MS/MS in negative ion mode. I am looking for a suitable internal standard, however, as expected, other fatty acids commercially available are subject to interference by endogenous levels or other similar compounds. I wondered, before I purchase, whether isotope labelled fatty acids e.g. stearic acid-13C or linoleic acid-13C, are sufficiently different in mass so that the mass transitions are free of interference from the corresponding unlabelled compounds or other similar mass compounds. Would appreciate feedback from those with experience.

Hi,

We use a compound available from Cambridge Biosciences, a d5 deuterated fatty acid that works very well for our analysis, which is the same method that you are using.

I haven't worked with fatty acids specifically, but I have used deuterium labeled versions of the analyte as an internal standard in pesticide and pharmaceutical type stuff. It's always worked quite well.

Edited to add: Usually the only reason not to use them is they can be quite expensive.

You could try a longer chain fatty acid that is non-endogenous or something. What kind of gradient are you using?

Thanks to each of you for your replies. We use stable isotope labelled (SIL) compounds as internal standards if we can get them. In this case I am quantifying a fatty acid derivative of C18 length. Stearic acid seems to be in anything we inject and so is not suitable as an internal standard. I have seen commercially available SIL versions of stearic acid varying in the number of deuteriums incorporated into the molecule. My concern is that if I purchase one, say an M+1 or an M+3 version, and as you say they are expensive, it too will prove unsuitable due to the appeareance of peaks at the same retention time coming from similar size endogenous fatty acids. Currently I am using a completely unrelated compound, which works in -ve ion mode, as IS. It is reasonable but not ideal. I guess my questions are:
1. If I use SIL-stearic acid, what size mass differential would be sufficient to avoid interference; M+1, M+2 etc ?
2. The d5 fatty acid from Cambridge Biosciences - is it a labelled version of an endogenous fatty acid ? Which one ?
3. Any suggestions for synthetic fatty acids/derivatives of C18 or similar chain length ?

Chromatographic conditions: Column 50 x 2.1mm C18, 5uM particles.
Mobile phase A: 10mM ammonium bicarbonate pH 8.5
Mobile phase B: Methanol
Flow rate 0.3mL/min
Gradient 50% B to 90% B over two mins
plasma sample extraction: SLE

Ian

Wow you run the gradient in two minutes? Seems fast, but I am not an expert at these things. You could look into buying arachidic acid (C20) if its not endogenous.

I'm not really a fatty acid person, but I thought the C17, C19 odd chain lengths were rare in nature, and yet can still be purchased?

Call Nu-chek Prep http://www.nu-chekprep.com/p.htm

Yeah, the odds are rarer but you can buy them all - we have them all although we bought them while in Germany before we moved the lab to Canada. But I assume you can buy them wherever.

I developed a stable isotope tag to perform fatty acid quantification by MALDI-MS-MS. So we created our own standards by differential labeling (with a light an a heavy tag).

I'm sure this derivatives will also work with HPLC-MS (the tag will also increase the ionization efficacy of ESI because of a quarternary amine)

If you are interested you can find the work here http://pubs.acs.org/doi/abs/10.1021/ac900562j

or write me a pm, I may have some spare label in the freezer.

cheers,

sirdaniel