A highly efficient method for determination of some amino acids and glutathione by liquid chromatography
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The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jabłonna, Poland
Publication date: 2003-01-02
J. Anim. Feed Sci. 2003;12(1):199-215
High-performance liquid chromatography systems (I and II) with pre-column derivatization for separation of cyst(e)ine, selenium-cystine, homo-cystine, methionine, selenium-methionine, proline and glutathione (GSH) in biological materials are described. Biological samples were derivatizated with o-phthaldialdehyde (OPA) in the presence of ethanethiol. Prior to derivatization, cyst(e)ine, methionine, GSH and proline were oxidized using improved procedures. Performic acid and sodium hypochlorite were used as the oxidizing reagents. HPLC analyses of derivatives of all oxidized compounds were carried out using a C18 column (4 µm, 250 x 4.6 mm I.D., Nova Pak, Waters) and binary gradient elution program I (HPLC system I). HPLC system I with UV monitoring (at 336 nm) and fluorescence detection (excitation and emission wavelengths at λexem= 336/425 nm) was chosen as providing the optimum conditions for fractionation and quantification of all of the examined compounds. In comparison with the UV detection, fluorescence detection offers better sensitivity (limits of detection:0.9-2.4 vs 0.3-0.8 ng⋅l-1). Clear separation of cyst(e)ine, GSH, methionine and proline was obtained in about 35 min. Separation of unoxidized GSH, cystine, seleno-cystine, methionine, seleno-methionine, homo-cystine from other free amino acids was achieved using HPLC system II with UV detection at 337 nm and/or fluorescence detection (λexem=336/425 nm). Compared with fluorescence detection, UV monitoring offers better limits of detection (LD) of cystine, seleno-cystine and homo-cystine, while worse L D for GSH, methionine and seleno-methionine. The satisfactory purity of analytical peaks of the assayed compounds (near 100%), and the simplicity and precision of HPLC systems I and II render these methods suitable for routine analysis of these compounds in large numbers of biological samples.
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