Determination of acylcarnitines in urine of patients with inborn errors of metabolism using high-performance liquid chromatography after derivatization with 4'-bromophenacylbromide.
A high-performance liquid chromatographic method is presented for the determination of urinary acylcarnitines. After solid phase extraction on silica columns the acylcarnitines are converted to 4'-bromophenacyl esters with 4'-bromophenacylbromide in the presence of N,N-diisopropylethylamine. Complete derivatization was achieved at 37 degrees C within 30 min. The 4'-bromophenacyl esters were separated by high-performance liquid chromatography on a Hypersil BDS C8 reversed-phase column with a binary gradient containing varying proportions of acetonitrile, water and 0.1 M triethylamine phosphate buffer. Essentially baseline separation was obtained with a standard mixture containing 4'-bromophenacyl esters of carnitine and synthetic acylcarnitines of increasing chain length ranging from acetyl- to palmitoylcarnitine. The method was used to obtain urinary acylcarnitine profiles from patients with propionic, methylmalonic and isovaleric acidemia and with medium-chain and multiple acyl-CoA dehydrogenase deficiency. Quantification of the acylcarnitines was achieved using undecanoylcarnitine as internal standard.
Poorthuis
B J
BJ
Department of Pediatrics, University Hospital, Leiden, The Netherlands.
Jille-Vlcková
T
T
Onkenhout
W
W
D000098:Acetophenones / Q000494:pharmacology*; D000108:Acetylcarnitine / Q000031:analogs & derivatives / Q000652:urine*; D044944:Acyl-CoA Dehydrogenases / Q000172:deficiency*; D000592:Amino Acid Metabolism, Inborn Errors / Q000652:urine*; D002331:Carnitine / Q000378:metabolism / Q000652:urine*; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid; D005021:Ethylamines; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D006801:Humans; D007223:Infant; D008052:Lipid Metabolism, Inborn Errors / Q000652:urine*; D008297:Male; D010741:Phospholipases A / Q000037:antagonists & inhibitors*
D000098:Acetophenones; D005021:Ethylamines; D005227:Fatty Acids; C084116:undecanoylcarnitine; C027070:N,N-diisopropylethylamine; D000108:Acetylcarnitine; D044944:Acyl-CoA Dehydrogenases; D010741:Phospholipases A; C006821:4-bromophenacyl bromide; D002331:Carnitine