Determination of homogentisic acid using HPLC with amperometric detection at carbon paste electrodes

Abstract

Homogentisic acid is an intermediate of the metabolic breakdown of tyrosine and phenylalanine; its accumulation in body fluids suggests the break in the metabolic pathway of these compounds and related health-threatening complications. In our work, a determination method for this compound was developed using HPLC with amperometric detection on glassy carbon paste electrode. Optimum conditions were found, considering both electrode response and chromatographic parameters. Separation was performed on Kromasil 100 C18, 250 x 4.6 mm (5 μm) column using gradient elution; mobile phase consisted of acidic buffer and methanol, with methanol content changing from 5% to 30% in 10 min. Repeatability of the measurements and linearity of the electrode response were confirmed. Applicability of the method was proved by the determination in spiked urine samples.