Plasma Amino-Acid Determinations by Reversed-Phase HPLC: Improvement of the Orthophthalaldehyde Method and Comparison with ion Exchange Chromatography

Abstract

Reversed-phase high performance liquid chromatographic (RP-HPLC) determination of amino-acids with on-line pre-column ortho-phthalaldehyde (OPA) derivatization and fluorescence detection is rapid and sensitive. However, high-performance ionexchange chromatography (HP-IEC) with post-column ninhydrine reaction is the most widely used amino-acid (AA) assay for biological samples. These two methods have been compared for the determination of individual plasma A A concentrations. An excellent correlation (p≤0-003) was found between the results given by an RP-HPLC system (Vartan) and a classical AA autoanalyser (Biotronik LC 5001). Most AA concentrations were similar with the two methods, but a paired t-test showed slight differences for 13 AA. The within-batch reproducibility of the RP-HPLC method was comparable to that of the HP-IEC method. The analysis was about three times faster with RP-HPLC’, and sensitivity was 100-fold better. However, aspartic acid, proline and cysteine were not identified by the RP-HPLC method, while the tryptophan quantification is possible. RP-HPLC with automated pre-column OP A derivatization is clearly a suitable alternative for assaying physiological AA and may be particularly useful for AA present at low concentrations (free tryptophan, plasma 3-methylhistidine). © 1992, Taylor & Francis Ltd.