Analysis of amino acids as formamidene butyl esters by electrospray ionization tandem mass spectrometry

Amino acid formamidene butyl esters are optimally prepared by heating amino acids with dimethylformamide dimethylacetal (DMF-DMA) for 2 minutes at 65 degreesC and then with n-butanol/hydrogen chloride for 15 minutes at 65 deg reesC. The formamidene butyl esters of simple ce-amino acids and beta -amin o acids afford between 1.1 and 20 times the signal intensity of the corresp onding butyl esters during electrospray ionization tandem mass spectral ana lysis. Complex alpha -amino acids, such as ornithine arginine and citrullin e, and gamma -amino acids are better analyzed as butyl esters. Secondary al pha -amino acids, such as proline and sarcosine, give a mixture of two imin ium salts with the DMF-DMA derivatization method. A derivative incorporatin g two molecules of the amino acid predominates at higher derivatization tem peratures. Contrary to a previous report, N-formylamino acids were not dete cted. The presence of secondary amino acids in amino acid mixtures, derivat ized as formamidene butyl esters, affords derivatives that incorporate two different amino acids. The new formamidene butylation method is unlikely to replace the butylation procedure used by neonatal blood spot screening pro grams for amino acid disorders, since a much poorer response was obtained w ith formamidene butyl esters of arginine and citrulline, important in the d iagnosis of arginase deficiency and citrullinaemia. Copyright (C) 2001 John Wiley & Sons, Ltd.