Chromatography Forum

Is this a potential LC/MS application or am I best advised to try another tack ?

That's a definite "maybe". A simple "mass detector" (usually a single quadrupole instrument) should work, but there are several potential problems:

- if your "typical reverse phase C18 setup" uses something like a phosphate buffer, you will have to re-work the separation (non-volatile buffers are a problem; some of the newer systems will tolerate them for short periods of time, but they are best avoided)
- a very complex sample like an herbal extract have other junk with an m/z of 441 eluting near your folic acid (and I'm not enough of a mass spectrometrist to tell how well folic acid will be ionized and detected).

Before spending a lot of time or money, I would talk to your friendly local LC-MS sales person (from Thermo, Waters, Agilent, Shimadzu, and whomever else I'm missing) and see if they would run a test sample for you to evaluate feasibility.

You can almost certainly do it via LC-MS/MS using a triple quadrupole system. These are routinely used for bioanalytical work looking at very low levels of drugs or metabolites in biological fluids or tissues. You end up looking at specific fragments of specific molecules, so you can deal with the very complex samples. The catch is, they ain't cheap (you're looking at >$200k).

Need to also consider matrix ionization effects. Even though the MS/MS gives specificity, minor components of interest eluting with larger components can still be problematic and lead to errors in quantitative analyses.

Good book:

http://www.amazon.com/gp/reader/0849319 ... eader-link

some info on my web site: http://users.chartertn.net/slittle first section

It's been a while since I analysed some folate compounds, but I think the literature that I read suggested that Folic Acid is often determined using fluoresecene detection, are you able to use that?.

The other possibility may be to remove some of the offending junk using a precleaning step, such as SPE, or extraction, but the ease may depend on your sample. I'd suggest a WWW search using suitable keywords in case there are methods around.

Please keep having fun,

Bruce Hamilton

Have you considered SPE with an technique orthogonal to reverse phase? Maybe you can use anion exchange SPE to remove the non-acid species. You may still have too many interferences, but it could be worth trying before you buy a new instrument. Also, you may still need to do this even with LC-MS.

I am fairly sure that you can get a rather clean extract with SPE. How good the cleanup will be, depends on the sophistication of the SPE procedure that you are willing to use. My suggestion is to use a standard mixed-mode anion exchange procedure first, which will allow you to remove ALL neutral and cationic interferences. In most cases that I have encountered, this is rather sufficient to get good results. However, a higher sophistication can be included to get an even better cleanup. I suggest you look at the protocols on the Waters webside for Oasis MAX. Here is a brief summary: put your analyte into the anionic form, typically done by addition of ammonia. Load it onto an Oasis MAX cartridge. Wash basic and neutral interferences away with methanol and some ammonia. Elute your analyte with formic acid in methanol.

You can try to use mixed mode approach without any sample prep. Neutral, acidic and basic compounds can be moved independently by variation of buffer concentration, buffer pH and amount of ACN:

http://www.sielc.com/Technology_2D_Properties.html

Here is analysis of caffeine in coffee. Coffee has a lot of other "junk" besides caffeine:

http://www.sielc.com/application_174.html


If you send us sample we will show you how this can be achieved (at no charge).

Regards,

Vlad

Of course we are interested.
It seems, though, that many people are too embarassed to post, once they found solutions to their problems?

I was wondering if you are using uv detection, would it be possible to make the detector more selective by choosing the right wavelength.

GCguy

DawnRazor,

I would love to hear about the solution. I too wonder what really did happen, especially on threads where I cannot offer any advice.

Best regards.

Hello, I think SPE is a good way to clean up the sample and the procedure decribed by Uwe is very good I tyried already with folicacid on fortified rice and it was good, still I have some issues with this procedure, but I have some information that could be usefull because they analyze Hydrosoluble vitamins in very complex samples.

I can guive you a copy of this article, just let me know.


Oscar Cortes.