Chromatography Forum

hi,

i'm trying to develop a method to analyse amino acids without derivatization on LC-MS/MS but i have troubles to get a good seperation.

anybody has any solution on this problem?

Best regards

Hello willemdm

Well, the separación is important in the mass spec, but if your column or mobile phase can't go further, you have to separate each aminoacids in different chanels , in the MRM function.
In this way, it won't matter the overlaping Because you ll get all your peaks by separate, just be sure to add the correct channel in the moment you want to quantify your data.
A mobile phase could help you is trifluoroacetic acid 0.1 % in water and ACN as weel.
I have some methods for running aminoacids without derivatization, let me know if you need it.
Another question, you are running aminoacids from a protein hydrolisis in the food, or just added aminoacids?

If several compounds co-elute, there is a risk of ion suppression which may lead to inaccurate results. Separation is still important otherwise we would all use direct infusion MS/MS and not bother with chromatography anymore

Have you tried on a C18 column? You may use HILIC as an alternative to derivatization, there are quite a few protocols published in the literature.

I would avoid TFA in case your instrument is used for various applications including in negative ion mode. TFA causes strong ion suppression in neg mode and is difficult to remove from the system.

Hello--feel free to review this...don't know if it will work for MS/MS, but it's a nice paper. Don't need to use TFA for this...see what you think.

http://www.sciencedirect.com/science/ar ... 7399008638

Journal of Chromatography A, Volume 870, Issues 1–2, 18 February 2000, Pages 245–254

Ion-pair chromatography on a porous graphitic carbon stationary phase for the analysis of twenty underivatized protein amino acids P Chaimbault, K Petritis, C Elfakir, M Dreux

Institut de Chimie Organique et Analytique (IC.O.A), CNRS UPRES-A 6005, Université d’Orléans, BP 6759, 45067 Orléans Cedex 2, France

Abstract

The analysis of twenty underivatized protein amino acids has been achieved on porous graphitic carbon packing material (Hypercarb). Five perfluoroalkyl carboxylic acids (trifluoroacetic, heptafluorobutyric, nonafluoropentanoic, tridecafluoroheptanoic and pentadecafluorooctanoic acid) have been studied as ion-pairing reagent. Several parameters (equilibration time, quantities adsorbed onto the chromatographic support, concentration and nature of the ion-pairing reagent, as well as temperature effect) have been studied leading to the complete separation of these compounds in gradient elution mode. Evaporative light scattering detector has allowed the detection of these non UV–visible absorbent molecules. The chromatographic methodology developed can also be easily coupled with pneumatically assisted electrospray mass spectrometry

Especially See Fig. 5. LC–ELSD analysis of the 20 underivatized amino acids in gradient elution on Hypercarb (100×2.1 mm I.D.). Eluent A: nonafluoropentanoic acid (NFPA) 20 mM in an aqueous mobile phase. Eluent B: Acetonitrile. Gradient profile is: from 0 to 15% ACN in 10 min, then 26% ACN in 10 min and finally 50% ACN in a further 10 min. 50% ACN is maintained until the end. Column temperature is 10°C. Flow rate: 200 μl min−1. Detection: Evaporative Light Scattering Detector (ELSD) set as in section 2.2. Imp: Impurity

Matt

I agree that a hypercarb has a good chance of retaining and separating, but reproduceability can be a problem. I have found that the column can take a long time to recover from pH and ionic strength changes in mobile phase (going from nuetral to acidic pH and then back again may give weird results when "back again").

You could also try silica, cyano, diol, PFP, or amino columns with reverse phase mobile phases.

Good luck.

Alp

Good Morning,

Links to a couple of other possible methods:

http://www.chromatographyonline.com/lcg ... ail/739811

I've used the Thermo Dionex Acclaim RSLC PAII for another application or two...solid performance for explosives and DNPH-derivatized carbonyls.

http://www.ucdenver.edu/academics/colle ... strong.pdf

This was nice as the entire paper is here and available to read for my favorite price...free. I've also used this particular Agilent column type in the past...solid performance, but again, not with the amino acid separation, but for silybins.

Can't say I've used Hypercarb myself, I respect Alp's opinion...just sending out references. With best luck, one will work out...at least I hope so.

Was thinking of Hypercarb as a means of separating the E/Z isomers created in derivatizing carbonyls with DNPH...maybe that idea isn't such a hot one if Hypercarb has reproducibility troubles...my thanks to Alp for sending along the opinion!

Not trying to trash the graphitic column (hypercarb) I did develop a method that passed validation on this type of column for urea by lcms ms. I was able to retain the analyte outside the void volume. NOTHING else worked (this was pre-HILIC).

The hypercarb CAN do wonders but you have to be aware that if you change mobilephase things up enough it can start to act very strangely for a hell of a lot longer than you would expect a similar silica based (c-18, cyano, amino...) to recondition to new mobile phase.

Alp

Hi Alp,

No worries on my account...just spinning references. You do a very proper thing by accounting your experience with the Hypercarb phase...I appreciate your comment.

As in most things in life...something has to be tried out to see how it will or will not do. Was glad to find a couple of references with column phases I've worked with that may be less problematic with non-derivatized amino acids (Sergio and Gaetan have other ideas yet)...in any case, the experimental results will decide. All the rest, just a means to decide what experiment may be the best to try out first with a reasonable shot at success.

My thanks to everyone here...it's nice to talk about separations!

Matt

Any progress?

Andy

Is there a particular reason you are tying yourself to LC-MS? Phenomenex makes a kit that renders amino acids determinable by GC (part number KGO-7165). Pretty much all detectors apply.

also, just in case anyone does come across this thread, and tries to do it in the future:
I've tried something similar: nonofluoropentanoic acid as ion-pair reagent for amino acids, in my case just with a typical C18 column. The method worked quite nicely. I even moved it from one LC-MS to another.

But I couldn't use either LC-MS in negative mode for over a month afterwards, despite copious flushings of the entire LC system, replacing parts of the autosampler of one system, and completely rebuilding the ESI probes on both systems (normal spray-chamber cleaning having barely made a difference). Nonofluoropentanoic acid ionises phenomenally well, and sticks to almost everything in a mass spec, giving a huge signal in negative mode.

It's nice to avoid derivatising. It's also nice to keep your instruments in a usable state...

I do several amino acids via GC-FID. I use a modified ethyl chloroformate derivitization procedure. It can do every amino acid except arginine and I can do the sample prep in literally less than 20 minutes. I have't found any good way to do arginine though. Arginine doesn't react with with the alkyl chloroformates and I believe I read silylation gives a mix of tripple and quadruple derivitized analytes. Cystine is also a bit tricky as it is late eluting, had low response, and differing solubility than most amino acids but it is a dipeptide not an amino acid.