HPLC with electrochemical detection for determining homogentisic acid and its application to urine from rats fed tyrosine-enriched food.

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 +0.5 V vs. Ag/AgCl. The peak heights of HGA showed linearity (r = 0.999) ranging from 4.2 ng/mL to 168 ng/mL, and the detection limit was 1.2 ng/mL (signal-to-noise ratio, S/N = 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 (n = 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 0.1 mL of a rat urine specimen and simple sample preparation consisting of dilution and filtration.

Alkaptonuria.

A 69-year-old woman presented with a 30-year history of lower back and large joint pain of the hips and shoulders. On examination blue-grey, pigmented macules were present over the cartilaginous portions of the ears and on the sclera. Past medical history included aortic stenosis. Urine homogentisic acid level was elevated, which is diagnostic for alkaptonuria. Alkaptonuria is an autosomal recessive disorder that results in deficiency of homogentisic acid oxidase and in the accumulation of homogentisic acid in connective tissue. Disease can result in blue-grey pigmentation of the cartilage, sclerae, face, and hands as well as severe arthropathy and cardiac valve disease. Treatment is limited at this time. Promising early reports of the use of nitisinone have prompted ongoing trials of this therapeutic agent.