Analysis of aspartylglucosamine at the picomole level by high-performance liquid chromatography

Abstract

A sensitive method for quantitative analysis of aspartylglucosamine as its dabsyl chloride derivative by high-performance liquid chromatography is described. Precolumn-derivatized aspartylglucosamine and internal standard (carboxymethylcysteine) are separated on a reversed-phase column with a mobile phase consisting of phosphate buffer and acetonitrile and monitored by UV-VIS detection at 436 nm. Aspartylglucosamine acts in the assay like a polar amino acid, and it can be separated from interfering substances in urine with a retention time of ca. 13 min. Its detection limit is ca. 0.3 microM in water and 0.5-1.0 microM in urine and other biological samples, which permits its quantitation in normal urine, for example. The within-day coefficient of variation is less than 4.7% and the day-to-day coefficient of variation is less than 8.3%. The present method is applicable to the direct analysis of aspartylglucosamine in body fluids and tissues without any prepurification and, in combination with automated liquid chromatography, allows rapid assay of a large number of samples in the detection of aspartylglycosaminuria. The sensitivity of the assay also allows direct quantitation of aspartylglucosamine in normal urine and leukocytes of aspartylglycosaminuria patients, and may thus be used in metabolic studies of the compound.