High-performance liquid chromatography of amino acids in urine and cerebrospinal fluid

Abstract

Two different methods for analyzing amino acids by reversed-phase high-performance liquid chromatography (HPLC), both of which can separate d- and l- stereoisomers, have been used for studying the amino acid composition of cerebrospinal fluid (CSF) and urine. One method, by which Dns derivatives of amino acids are separated as mixed chelate complexes with Cu(II) and a single stereoisomer of a second amino acid, was used to analyze CSF. CSF contains ca. 10 μmole/l per amino acid, compared to 100 μmole/l in serum. The high sensitivity of fluorescence detection enabled complete analysis, starting with 50 μl of fluid. The second method, which uses lower concentrations of both the copper and the second amino acid and detects amino acids by the change in absorbance of the copper complex, was used to measure the urine concentration of the lysine metabolite, pipecolic acid (piperidine-2-carboxylic acid), a secondary amino acid that is difficult to detect by the more usual detection methods. Our procedure involves passing urine through a cation-exchange column, collecting the fraction containing pipecoli acid, and chromatographing it on reversed-phase HPLC column with a mobile phase containing l-aspartame and Cu(II). To asses the utility of the method, urine samples from a patient given loading doses of d- or l-isomers were analyzed. When either isomer was administered, both d- and l-isomers were detected, but in different proportions. Varying proportions and concentrations of both isomers were also detected in the urines of patients with hyperpipecolatemia from different metabolic abnormalities.