Hi all!
Typically we have been using a C18 Reverse Phase column for our studies, unfortunately there is no retention of most polar metabolites and all elute at very early retention times. This is, unfortunately, a well known problem that may cause problems of ion supression.
I know there is not any analytical technique than can provide the whole metabolome profile of an organisms and typically both LC-MS and GC-MS are used. However, I would like to know your past experiences with other LC columns obtaining "most" of the information of metabolomes with just LC-MS. I know that T3 RP or HILIC columns may help with this issue but I never tried before... It is a bit frustrating the companies offer a huge variety of columns but there is no publications of robust methods for LC-MS plant metabolomics comparing deeply different columns and chromatographic methods. I suppose that it is a huge effort (economically and timing!). Plant extracts, contrary to human fluids, typically present a very complex matrix of compounds. I used to extract the metabolites with MeOH/H2O (80:20) which extract several polar and semi-polar metabolites. I am afraid that a HILIC column may no retain most of the semi-polar metabolites.
Any thoughts?
Thanks in advance!
http://www.mdpi.com/1420-3049/18/1/244
My paper describes method for polar extract, and I made SPE of my extracts.
my dissertation
http://www2.fcfar.unesp.br/Home/Pos-graduacao/CienciasFarmaceuticas/fabiana-volpe-zanutto---me.pdf
Good luck!
Hi Albert,
in the field of water and wastewater analysis we had the same problem. A possible solution for you could be a short PGC (Porous graphitic carbon) pre-column.
Here an example: http://link.springer.com/article/10.1007/s10337-014-2789-3
Good luck...
I see two ways to solve this problem. First, you could try another column (Raptor Bi-phenyl column - for Increasing retention of early-eluting compounds). Second way, as Fabiana has recommended - enhanced clean-up (may be DLMME instead SPE as additional step).
I would try a mixed-mode RP. Most polar metabolites would be possibly ionic at a certain pH. Even if they are not retained by RP, they will get retention by ion-exchange.
PGC is interesting if you have mostly polar compounds, but it is hard to get apolar compounds off the column. If you need extra separation power, I would couple mixed-mode RP with a PFP or use a phenyl-SCX as column. This will give reversed phase retention (not as much as C18, though), cation-exchange as extra retention mechanism and pi-pi interactions to better separate phenyl containing groups.
yes, but you only have one separation mechanism and it is hard to control it because of the fierce hydrophobic interactions.
I prefer a more subtle approach, with much more possibilities to tweak the method. :-)
Thank you all for your suggestions!
I will need to do several tests before decide anything. I see many possibilities...
Everybody suggest to use the GC-MS for the most polar metabolites, however, it is not always possible and I would like to obtain a fair range of polar metabolites with LC-MS as well without lossing the "semi-polar"ones.
I will keep you updated if I found a good strategy during the following weeks.
Thanks again!
You might try to do two LC of the same extract using orthogonal columns/methods, say an HILIC and an RP or two RP columns like a standard inactivated C18 an a more polar one like a ciano or diol. After that you might combine the two set of data obtained. At least in theory you should cover more compounds there. However is useful some sample prep to take away the part of sample you don't analyse and some pH adjusting.
For extraction of plant metabolome use this highly recommended Oliver Fiehn's protocol: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2007.03387.x/full
MeOH-Water works for phenolics, flavonoids, steroids but would be limited for sugars etc.
Also, C18 works perfect for most compounds if you are using MRM or targeted methods. I could get 350+ metabolites of metabolism interest in one run. Masami Hirai/ RIKEN have almost obtained 600+ compounds using MRM methods.
HILIC has limited applications in metabolomics due to peak shifts etc, and hence, if not well established not worth trying or reproducible.
LC-MS combined to GC-MS is preferred. GC-MS is highly recommended for sugars, TCA cycle intermediates, steroids and semivolatiles as well.
A combined apporach will help for sure.
Thanks a lot,
Biswa
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