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 primar y and secondary amino acids included cystine and homocystine in urine samples is described, After a simple ultrafiltration, urine samples we re subjected to reduction of disulfides, carboxymethylation of free th iols and double precolumn derivatization with o-phthalaldehyde-3-merca ptopropionic acid and 9-fluorenylmethyl chloroformate. All reactions w ere fully automated by means of an injector programme and were accompl ished in 10 min. Since urine samples contain a large number of amino c ompounds, a good resolution was required, By optimization of the condi tions, separation of 40 amino acids in 92 min was achieved. The recove ry 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 metab olism.