A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide:  Elucidation of Limitations of Marfey's Method and of Its Separation Mechanism

Abstract

As the first step in establishing our proposed method, the advanced Marfey's method, which is planned for the simultaneous determination of the absolute configuration of amino acids in a peptide, we applied Marfey's method to commercially available amino acids, and the separation behavior was examined in detail. Although good resolution of the diastereomeric pair of an individual amino acid was obtained for all amino acids tested and the applicability of the method was confirmed, the (1-fluoro-2,4-dinitrophenyl)-5-l-alaninamide (FDAA) derivative of the l-amino acid was not always eluted prior to its corresponding d-amino acid derivative. Because this proposed method relies on the elution order of a derivatized amino acid with FDAA to determine its absolute configuration, its separation mechanism was carefully investigated using UV and NMR spectral techniques. The results suggested that the resulting conformations of the l- and d-amino acid derivatives are stable and that the resolution between the l- and d-amino acid derivatives is due to the difference in their hydrophobicity, which is derived from the cis- or trans-type arrangement of two more hydrophobic substituents at both α-carbons of an amino acid and l-alanine amide, so that the FDAA derivative of the cis (Z)-type arrangement interacts more strongly with ODS silica gel and has a longer retention time than that of the trans (E)-type arrangement. Therefore, the l-amino acid derivative is usually eluted from the column before its corresponding d-amino acid derivative in Marfey's method. According to this separation mechanism, the elution order of a desired amino acid can be elucidated from the average retention time of the l- and d-amino acid derivatives, and the dl-serine and -asparagine derivatives are critical for Marfey's method.