Automated method for the measurement of amino acids in urine by high-performance liquid chromatography

An automatic and sensitive HPLC method for the determination of primary and secondary amino acids included cystine and homocystine in urine samples is described. After a simple ultrafiltration, urine samples were subjected to reduction of disulfides, carboxymethylation of free thiols and double precolumn derivatization with o-phthalaldehyde-3-mercaptopropionic acid and 9-fluroenylmethyl chloroformate. All reactions were fully automated by means of an injector programme and were accomplished in 10 min. Since urine samples contain a large number of amino compounds, a good resolution was required. By optimization of the conditions, separation of 40 amino acids in 92 min was achieved. The recovery of amino acids ranged from 83% for TRP to 105% for CIT. The within-run and between-run R.S.D.s of urinary amino acid concentrations were below 10% for most amino acids except for HYL, LYS and ORN. The method was applied to the diagnosis of genetic disorders of amino acid metabolism.