Instability of orthophthalaldehyde reagent for amino acid analysis

Determination of amino acids by reversed-phase chromatography of the adduct with orthophthalaldehyde and a thiol is rapid and sensitive. The major recognized adverse feature of this method is the instability of the reaction product, which requires precise control of reaction timing and chromatographic parameters for reliable quantitative application. We report another source of major variability: reagent instability. Deterioration of reagent was noted as low peak heights and peak broadening and was predictable if the premixed reagent was left at room temperature. Restoration of sharp chromatograms was accomplished by addition of mercaptoethanol or sodium metabisulfite. Reagent which was chromatographically inert contained minimal free thiol by direct assay. Free thiol disappearance was markedly slowed by addition of a chelating agent. Excess mercaptoethanol was deleterious. We conclude that reagent deterioration represents oxidation of the thiol, may be reversed by rereduction with minimal thiol or bisulfite, and may be minimized by inclusion of a metal chelator in the reagent.