Issue with amino acid analysis

[zilal]

Hello. I've been using a Waters UPLC to analyze amino acids; we've chosen FMOC for the derivatizing agent because we'd like to be able to cleave it from the AAs and put just the AAs through a mass spec for isotope analysis later on. The problem is that we haven't been seeing any peaks for amino acids in the chromatograms; just some small peaks in the first minute and some larger ones in the last minute of the 10-minute runs, the size of which corresponds to how much FMOC was added during derivatization, but nothing in between. So far, Waters' suggestion is to use Accq-tag, but we'd really like to make the FMOC work.

If this issue is up anyone's alley, let me know and I'll go on to bore you with some figures on our eluents, gradients, and derivatization procedures.

[mbulentcakar]

Hi Zilal,
I'm pretty sure many of the posters of the board have experience about the AA analysis.
I'd suggest you to put in more details about the procedure and the problem you're facing.
Regards,
Bulent

[HW Mueller]

You can get commercial FMOC derivatives of lots of amino acids to set up your LC analysis.

[zilal]

Okay... we've tried lots of stuff, so there's some variation in our methods, but in the end none of it worked. For derivatization, we'd been putting the amino acid standards into a sodium borate buffer that ranged between 0.1 and 0.5 M. FMOC was dissolved in either acetone or acetonitrile at 1 mM or higher concentrations, and added to the amino acid standard at a molar ratio of AA:FMOC of 1:10 to 1:50. The reaction always had at least half an hour to sit, usually a few hours or a day. We haven't measured the actual pH under which the derivatizations have progressed. They've been at room temperature.

Samples were then either injected directly or dried under nitrogen gas and redissolved in methanol before injection. Our formula for Eluent A has approximated that used in the Accq-Tag kit; a concentrate of 190g sodium acetate added to 1 L of water, which was then titrated to either pH 5.05 or 4.2, and diluted 10x before use. We've tried it with TEA and without TEA. Eluent B is 100% methanol, ACN still being a bit pricey. Strong wash is 100% MeOH, weak wash is 10% MeOH in water.

The gradient we've been using comes from some Waters literature on using the UPLC to analyze amino acids.

Time % A curve
Initial 99.9 6
0.54 99.9 7
5.74 90.9 6
7.74 78.8 6
8.14 40.4 6
8.25 10.0 6
8.93 99.9 6

I don't recall the name of the column offhand, but it was whatever Waters recommended for AA analysis. It's being run at 60 degrees C, with the UV detector set to 260 nm.

I've seen the pre-derivatized FMOC-AAs for sale and am interested in trying them, but my boss is sure we're doing the derivatization wrong and don't need to spend more money to confirm it. Any insight on our lack of peaks is appreciated!

[HW Mueller]

Do I understand this? You are using the Waters LC method which is not developed for FMOC derivatives?

[zilal]

Do I understand this? You are using the Waters LC method which is not developed for FMOC derivatives?

That's quite right. We have lots of papers on hand in which the researchers used FMOC with an HPLC, from which we've gotten many of the variations in method we've tried, but none with a UPLC.

[skunked_once]

Do you have access to a standard HPLC so that you can duplicate the FMOC analysis in your references? If you can't get a decent standard run on a conventional HPLC, the problem may be in your derivatization. If your derivatized standards give a good chromatogram on the conventional HPLC, the problem is in transferring the method to the UPLC. Sorry that I can't help you with that but the manufacturer should be able to give you some advice.

[skunked_once]

That's quite right. We have lots of papers on hand in which the researchers used FMOC with an HPLC, from which we've gotten many of the variations in method we've tried, but none with a UPLC.

Is the gradient you're trying obtained from the FMOC references? If you are using the Waters gradient that is set up for a different derivatization procedure, I'm not surprised that it doesn't work and wouldn't expect it to work.

[Hollow]

Maybe a silly question:

How about the scaling of the y-axis?

If it's autoscaled, then you probably will see only the excess of the FMOC and your real AAs are just as tiny and be overlooked?

BTW:
Keep in mind that the gradient you're using was developed for the ACC-tag and not for the FMOC (if I'd understood it right). It's pretty flat with just some "cleaning" step at the end.
Try some standard gradient from 0-100% to see in which region the FMOC-derivatives elutes.
Maybe there are some gradients for FMOC published in the literature. Try to use one of them, correctly scaled to the UPLC system.

[mbulentcakar]

Hi,
As far as i understand, your first aim is to make sure derivatization is ok.
If this were my problem, I would try to derivatize AA standards with FMOC, extract with pentane and shoot into hplc without column (maybe after evaporation and dilution with your intented mobile phase) using hplc as a spectrophotometer. After optimizing derivatization, i would go on hplc, or uplc method development/optimization.
Best regards,
Bulent

[HW Mueller]

Bulent, that isn´t going to work. FMOC-Cl and FMOC derivatives have the same UV and fluorescence spectra.

zilal, the FMOC-AA standards are more important for setting up your HPLC than the derivatization. FMOC-CL is very reactive, it is almost impossible not to have it react with amino acids. The main drawback is rather that it also reacts with water, NH3 (it makes the traces of NH3 visible that come with ~ all amino acid preps.), and that reagent fluoresces. So once your HPLC is running you just optimize (mostly temp., time; the pH is known from the lit.) the reaction to minimize reaction with H2O etc. You get rid of excess FMOC-Cl via a polar amino acid (if you want the excess to come out ahead of your AA analytes) or amino-adamantane (if it is more convenient to have the excess come out after your amino acids). You may need a bit of a reoptimization of your HPLC at this point to separate the H2O and NH3 derivatives.
Now this has been mentioned before and is available from all kinds of other sources.

Incidentally, in our hands, the FMOC method proved far superior to a number of other derivatization methods, some reasons have been mentioned earlier.

[mbicking]

Agilent has a method that uses OPA to do the primary AA's, and FMOC for the seconary ones (after initial addition of OPA). The reactions are rapid and done in the autosampler. A borate buffer is also used, suggesting that the chemistry is similar.

The method instructs you to change the OPA and FMOC daily, due to instability. Is it possible that your reagents are not active?

[mbulentcakar]

Bulent, that isn´t going to work. FMOC-Cl and FMOC derivatives have the same UV and fluorescence spectra.

Hi Mr. Mueller,
That's why i also suggested the pentane extraction. Not very efficient and cumbersome (4-5 times liquid-liquid extraction) but aids to get rid of most of nonreacted FMOC and also FMOC-OH. Others have used a secondary derivatization for this purpose, which is said to be more efficient but i have no experience on that.
Best regards,
Bulent

[DWR]

Hi ZILAL
This may be of interest: Waters had some information for lc ms/ms of fmoc amino acids a few years back. Derivatisation was equal volumes of sample, fmoc in acetonitrile, ammonium acetate/NH4OH. Mobile phases were A)10mm NH4OH B)Acetonitrile with an XTERRA column. Negative esi.
We tried it but had a few problems with separating isobaric amino acids consistently. I am going to retry it for a small panel of amino acids. Obviously the excess reagent is not a problem with mass spec detection. Please contact me if you want further details of gradients, mass transitions etc.