ORIGINAL PAPER
Determination of free amino acids in blood plasma by high-performance liquid chromatography with fluorescence detection
 
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The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jabłonna, Poland
Publication date: 1998-10-22
 
J. Anim. Feed Sci. 1998;7(4):453–463
 
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ABSTRACT
A high-performance liquid chromatography method with pre-column derivatization for separation and quantification of free amino acids in blood plasma samples is described. Plasma samples after deproteinization with 5-sulphosalicylic acid are used for derivatization with o-phthaldialdehyde (OPA) in the presence 2-mercaptoethanol (reagent 1) or ethanethiol (reagent 2). Primary amino acid derivatives were separated on a C-18 Nova-Pak column (4 µm, 250 x 4.6 mm I.D., Waters) by gradient elution and fluorescence detection. Cystine, cysteine and proline are oxidized with sodium hypochlorite prior to derivatization with OPA in the presence of ethanethiol. Separations of derivatives of oxidized amino acids are carried out using a C-18 Symmetry column (5 µm, 250 x 4.6 mm I.D., Waters) by gradient elution and fluorescence detection. All OPA amino acid derivatives are detected with a monochromator set at 338 nm with a 425 nm cut-off filter. Clear resolution of at least seventeen amino acids and taurine was obtained in about 42 min. Low values of within and between run coefficients of variation, rapid derivatization and high sensitivity (in femtomole range) render this HPLC method suitable for routine free amino acid determinations in a large number of blood plasma samples.
ISSN:1230-1388