Electrochemical activity of o-phthalaldehyde—mercaptoethanol derivatives of amino acids : Application to high-performance liquid chromatographic determination of amino acids in plasma and other biological materials

Abstract

o-Phthalaldehyde—mercaptoethanol (OPA) reagent is used to increase the sensitivity of post-column detection of amino acids after chromatographic separation. OPA-amino acids themselves chromatograph well on reversed-phase high-performance liquid chromatographic (HPLC) columns and can be determined by fluorimetric detection, enabling OPA to be used as a pre-column derivatisation reagent. In this paper electrochemical detection (in the 0.4–0.7 V range) of OPA-amino acids following reversed-phase HPLC is described. Using fluorimetric and electrochemical detection in series, confirmation of the identities of the amino acids can be obtained in a single chromatographic run. Particular amino acids (e.g. basic amino acids) are active at the lower potentials, so that they can be selectively detected if required. Other amino acids and peptides whose OPA derivatives have little or no fluorescent activity are electroactive, permitting their detection by HPLC of their OPA derivatives. Application of this methodology to divers biological samples is illustrated. OPA derivatisation of amines is an example of a reaction in which the product is electroactive at a lower potential than the reactants. This type of reaction is likely to be particularly useful in extending the applications of electrochemical detection in HPLC.