Determination of Urinary Catecholamine Sulfoconjugate Isomers Utilyzing High-Performance Liquid Chromatography and Photo-induced Fluorogenic Reaction

概要

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A method for the determination of catecholamine-O-sulfate isomers (CA-S)was made by using high-performance liquid chromatography (HPLC) and newly developed photo-induced fluorogenic reaction. CA-S and 3, 4-dihydroxybenzylamine 3-O-sulfate (DHBA-3-S: an internal standard) were synthesized chemically and separated from each other by HPLC using silica gel column. They were found to become fluorescent compounds with ethylenediamine under UV-light. This reaction was applied to the post-column labelling of CA-S separated by HPLC for sensitive and selective detection of CA-S, and a special equipment was made with UV lamp and reaction coil of teflon tube inside of a temperature-controlled reaction box. With this system CA-S were separated and could be detected in the level of ten pmol. Urine samples were pre-treated with small ionexchange resin columns for the removal of interfering substances. The peaks corresponding to CA-S on the chromatogram obtained from a urine sample were identified by two ways; the elution patterns of CA-S in urine from Dowex 1 (AcO) column were coincident with those of the sample added with standard CA-S and with no irradiation in the post-column labelling reaction no peaks were detected in the elution time on HPLC corresponding to CA-S. CA-S in urine of healthy men were measured and the existences of all CA-S were certificated in the physiological level.
Japan Society of Clinical Chemistryの論文