Determination of homogentisic acid in urine for diagnosis of alcaptonuria: Capillary electrophoretic method optimization using experimental design

Abstract

Homogentisic acid (HGA) is a diagnostic metabolite that accumulates in the urine and tissues of patients with alkaptonuria which is a rare autosomal recessive disease. HGA is a specific metabolite in urine and serum, which is used for diagnosis of alkaptonuria. This study presents an inexpensive and easy capillary electrophoretic method for the quantitative determination of HGA in urine samples. The method was optimized using full factorial experimental design. The optimal separation electrolyte and separation voltage were revealed as 45mmol/L phosphate buffer at pH7.0 and 22kV, respectively. Under these conditions the presence of HGA was detected in 6min. Repeatability of migration times and corrected peak areas of HGA (as RSD) were 0.37 and 1.99, respectively. The detection limit was 0.56g/mL, 3 times of the average noise, and the quantification limit was 1.85g/mL, 10 times the average noise for HGA. Urine samples were directly injected to the capillary without any pretreatment step.