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Total free catecholamines assay by identification of its two functional groups and micro-flow injection chemiluminescence.

We have developed a novel method of assaying total free catecholamines using sulphuric acid-derivatized beads for extracting and identifying catecholamine (CA) on the surface, and assaying the peroxide produced from CA by chemiluminescence (CL). Current assay methods for CA by electrochemical determination, fluorescence and chemiluminescence need a time-consuming separation by high-performance liquid chromatography. We eliminated this separation step by identifying the two functional groups of CA using a derivatized bead and this resulted in a highly specific CA assay. The principle is as follows: the amino group of CA was trapped by ion binding with a sulphuric acid derivative immobilized on a bead, and the diol of the CA bound to the bead was converted to peroxide with imidazole under alkaline conditions. The peroxide produced was assayed by microflow injection-horseradish peroxidase-catalysed luminol chemiluminescence. We synthesized three types of sulphuric acid-derivative immobilized beads (6.5 mm i.d.). The types of immobilized sulphuric acid derivative used were straight-chain, branched chain and benzenesulphonic, respectively. The order of the three types of beads for extracting CA was: bezenesulphonic type > branched type > straight-chain type. The optimal incubation time for generating peroxide was 30 min. The peroxide generated in the reaction solution was stable with within-run reproducibility of CV 5. 7% after incubation for 80 min. The regression equation of a standard curve for dopamine was Y = 12.8 X(2) + 476X - 373 (where Y = light intensity (RLU), X = concentration of dopamine (micromol/L)). The minimum detection limit of dopamine was 0.1 micromol/L, and the within-run reproducibility of dopamine (10.5 micromol/L) was CV 4.7% (n = 5). This method is applicable to assay of total free CA without use of HPLC.