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

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Abstract
Amino acid formamidene butyl esters are optimally prepared by heating amino acids with dimethylformamide dimethylacetal (DMF-DMA) for 2 minutes at 65 °C and then with n-butanol/hydrogen chloride for 15 minutes at 65 °C. The formamidene butyl esters of simple α-amino acids and β-amino acids afford between 1.1 and 20 times the signal intensity of the corresponding butyl esters during electrospray ionization tandem mass spectral analysis. Complex α-amino acids, such as ornithine, arginine and citrulline, and γ-amino acids are better analyzed as butyl esters. Secondary α-amino acids, such as proline and sarcosine, give a mixture of two iminium salts with the DMF-DMA derivatization method. A derivative incorporating two molecules of the amino acid predominates at higher derivatization temperatures. Contrary to a previous report, N-formylamino acids were not detected. The presence of secondary amino acids in amino acid mixtures, derivatized 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 programs for amino acid disorders, since a much poorer response was obtained with formamidene butyl esters of arginine and citrulline, important in the diagnosis of arginase deficiency and citrullinaemia. Copyright © 2001 John Wiley & Sons, Ltd.