Chromatography Forum

Hello,
I would like to strat an analysis of amino acids in human plasma. I did some researches, but i can not find a simple hplc method. I am working on HPLC-DAD detector and HPLC-Fluoresc.
Can you share with me your experience in this feild.

Thank you.

Free amino acids to determine there are some Special columns available on the market.
Standard methods use Pre- or Post-column derivatisation. Most column manufacturers will provide you with appropiate methods and columns.
Good luck.

Here's a simple method:

1) Add 2 vol. ACN to 1 vol. of plasma. This will precipitate any proteins or peptides > 6 KDa.

2) Inject the supernatant directly onto a PolyHYDROXYETHYL A column with a pore diameter of 100 Å, with isocratic elution with 15 mM ammonium acetate (pH 4.5) containing 60% ACN. Polar analytes will be retained via hydrophilic interaction.

3) Elute directly to a tandem mass spectrometer (MS/MS). Amino acids will elute between 2-4 column volumes past the void under these conditions and can be detected and quantitated.

This method is a combination of my poster from ABRF 2003, on the precipitation of proteins from serum with ACN, and of Robert Croes' (DuPont Biotech) posters from ASMS 1999 and 2000 on analysis of amino acids from individual kernals of corn or from individual soybeans. Let me know if you want copies of the posters in pdf format.

Andy Alpert
aalpert@polylc.com

Here are approaches using analysis of underivatized amino acids in various matrices:

Liquid chromatography/isotope ratio mass spectrometry measurement of δ13C of amino acids in plant proteins. Anthony H. Lynch, James S. O. McCullagh, Robert E. M. Hedges 2011 Rapid Communications in Mass Spectrometry, October 2011, Vol. 25, No. 20, p 2981-2988

Investigation of Amino Acid Signatures in Bone Collagen to Reconstruct Human Palaeodiets Using Liquid Chromatography-Isotope Ratio Mass Spectrometry Kyungcheol Choy, Colin I. Smith, Benjamin T. Fuller, Michael P. Richards, 2010 Geochimica et Cosmochimica Acta, 1 November 2010, Volume 74, Issue 21, Pages 6093-6111

Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl--quantification by ID LC-MS/MS. Lowenthal MS, Yen J, Bunk DM, Phinney KW. 2010 Journal of Chromatography A Volume 1217, Issue 37, 10 September 2010, Pages 5776-5784

Comparison of orthogonal liquid and gas chromatography-mass spectrometry platforms for the determination of amino acid concentrations in human plasma.
McGaw EA, Phinney KW, Lowenthal MS. 2010 J Chromatogr A. 2010 Sep 10;1217(37):5822-31. Epub 2010 Jul 21.

Direct analysis of underivatized asymmetric dimethylarginine (ADMA) and L-arginine from plasma using mixed-mode ion-exchange liquid chromatography-tendem mass spectrometry Michael J Bishop, Brian Crow, Dean Norton, Ekaterina Paliakov, Joe George, J A Bralley 2007 Chromatogr B Analyt Technol Biomed Life Sci. 2007 Oct 9;

A simple and selective method for the measurement of leucine and isoleucine from plasma using electrospray ionization tandem mass spectrometry Michael J. Bishop *, Brian Crow, Dean Norton, Kasey Kovalcik, Joe George, J. Alexander Bralley 2007 Rapid Communications in Mass Spectrometry, Volume 21, Issue 12 , Pages 1920 - 1924

Thank you very much for all your ansewrs.

I will try to look and search for methods with pre-derivatization using OPA as reagent, and DAD or Fluorescence Detector. I hope you can help me more to developping a method.

Thank you again.

Hey Andy,
I am running sports nutrition powders (includes whey protein among others) for free amino acids and caffeine. After derivatization with DNFB at pH 9, following bringing to pH 6 I filter through 0.2 micron nylon. I use a Brownlee C18 50x3 mm 2.7 um particle size. I believe the pore size is around 90 angstroms. I do not extract any proteins, and my diluent is water. Mobile phase is 84:16 10 mM phosphate buffer pH 6/acetonitrile. I sometimes see huge spikes in pressure. Usually the pressure reads 8100 psi at 0.7 ml/min (using a FX-15 perking elmer UHPLC). Is protein getting clogged in the column, especially when contact is made with ACN? I am freaking out.

Thanks so much
-Mike

Hi, Mike -

It's plausible that your whey proteins are precipitating and are clogging your column. Reversed-phase HPLC is not compatible with intact proteins in general. If you want to assess this for your sample, just dissolve these samples in water and then add 1 volume of ACN. If you see any cloudiness, then don't consider reversed-phase to be an appropriate mode unless you deproteinate your sample first. A convenient way to do that is to add 2 vol. ACN, spin down the precipitate, and then inject the supernatant directly (HILIC) or else collect it and dry it down for reversed-phase analysis.