Simultaneous high-performance liquid chromatographic determination of amino acids in a dried blood spot as a neonatal screening test

A new screening test on dried blood spots for inherited disorders of amino acid metabolism using reversed-phase high-performance liquid chromatography (RP-HPLC) is described. The method allows the simultaneous analysis of fourteen different amino acids; among these, seven whose blood levels are increased in the most important amino acid disorders have been determined. The procedure requires a preliminary extraction of the amino acids from 9-mm autoclaved dried blood spots by sonication in phosphate-buffered saline. A precolumn o-phthaldialdehyde-3-mercaptopropionic acid derivatization is then followed by analysis of the amino acids by RP-HPLC. Blood-spots levels of histidine (His), tyrosine (Tyr), valine (Val), methionine (Met), isoleucine (Ile), phenylalanine (Phe) and leucine (Leu) can be determined in a single 15-min run, including column washing and regeneration. The minimum detectable amount of each amino acid is 0.5 pmol with a linear dose-response range between 1 and 10 microM. The recovery for all amino acids is greater than 70% except for Met (66%). Up to 20,000 samples/year can be processed on a single automated analytical line resulting in an estimated cost of about US$ 0.25/sample. The multiple diagnostic capacity, the low cost and the possibility of complete automation of the method make it suitable for primary perinatal screening of amino acid disorders.