Automated high-performance liquid chromatographic determination of 5-S-cysteinyl-3,4-dihydroxyphenylalanine in urine

Abstract

An automated high-performance liquid chromatographic (HPLC) method has been developed for measurement of 5-S-cysteinyl-DOPA in urine (DOPA = 3,4-dihydroxyphenylalanine). The urinary sample was injected into an HPLC boronate column. With a mobile phase of 0.1 M phosphate buffer containing 0.2 mM disodium ethylenediaminetetraacetate (Na2EDTA) (pH 6.0) mixed with methanol (9:1), 5-S-cysteinyl-DOPA was adsorbed while most other compounds were washed away. By column switching, the column flow was reversed and 5-S-cysteinyl-DOPA was desorbed by a mobile phase of 0.1 M formic acid and 0.2 mM Na2EDTA at pH 3.0 and chromatographed on a reversed-phase column. The precision, as estimated from repeated analysis of an urinary sample and from duplicate analysis of a number of samples, ranged from 1.4 to 5.2% (coefficient of variation), and the analytical recovery was 93 +/- 4.1%. The method is suitable for use in the clinical laboratory.