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.
Department of Experimental Medicine, University La Sapienza, Rome, Italy.
F
F
Moretti
M
M
Birarelli
C
C
Carducci
A
A
Pontecorvi
I
I
Antonozzi
D000592:Amino Acid Metabolism, Inborn Errors / Q000175:diagnosis*; D000596:Amino Acids / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007231:Infant, Newborn; D009764:o-Phthalaldehyde
D000596:Amino Acids; D009764:o-Phthalaldehyde