RESUMO
A highly sensitive method for determining urine homogentisic acid (HGA) is required to provide adequate diagnosis and therapy for alkaptonuria in early stages. In this study, we developed a highly sensitive high-performance liquid chromatography with electrochemical detection (HPLC-ECD) for determining HGA in urine. In order to obtain a chromatogram of HGA by HPLC-ECD, an oxidation current was monitored at +0.5â¯V vs. Ag/AgCl. The peak heights of HGA showed linearity (râ¯=â¯0.999) ranging from 4.2â¯ng/mL to 168â¯ng/mL, and the detection limit was 1.2â¯ng/mL (signal-to-noise ratio, S/Nâ¯=â¯3). In recovery tests using human control urine spiked with an HGA standard, the recoveries of HGA were more than 93.2 %, and the relative standard deviations (nâ¯=â¯6) were less than 1.9 %. As an in vivo application using male Wistar rats, the level of urine HGA, which was metabolized from tyrosine in tyrosine-enriched food, was determined by this HPLC-ECD method. The determination of HGA in urine by this HPLC-ECD method requires only 0.1â¯mL of a rat urine specimen and simple sample preparation consisting of dilution and filtration.