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The "advanced Marfey's method" proposed in our preceding paper has been developed to nonempirically determine the absolute configuration of constituent amino acids in a peptide using liquid chromatography/mass spectrometry (LC/MS). For the establishment of this method, we had to resolve the following three problems: (1) elucidation of the limitation of Marfey's method, which is chosen as the chromatography technique, and its separation mechanism, because this proposed method relies on the elution order of an amino acid derivatized with 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (l-FDAA) to determine its absolute configuration; (2) how to effectively combine Marfey's method with mass spectrometry in order to detect and identify a desired amino acid without a standard sample; and (3) how to obtain the corresponding enantiomer from either the l- or d-amino acid in a peptide sample. In a preceding paper, we investigated problem 1 and finally described the rational application guideline for Marfey's method to elucidate the elution order of a desired amino acid according to the proposed separation mechanism. In this paper, we further investigated the two remaining problems. Because the sensitivity of the amino acids derivatized with the original derivatizing reagent, l-FDAA, was poor for LC/MS analysis using any interfaces due to their possible thermal instability and low hydrophobicity, we chose electrospray ionization and frit-fast atom bombardment (Frit-FAB) as the interface and developed 1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (l-FDLA) instead of l-FDAA as a new derivatizing reagent in order to combine Marfey's method with mass spectrometry. Furthermore, we introduced a racemization procedure using 1-fluoro-2,4-dinitrophenyl-5-dl-leucinamide (dl-FDLA), the "dl-FDLA derivatization", instead of the conventional chemical racemization for obtaining the corresponding enantiomer from either the l- or d-amino acid. Thus, we have established a nonempirical method using LC/MS, the "advanced Marfey's method". The method was successfully applied to the characterization of constituent amino acids in microcystin LR produced by cyanobacteria.
Fujii et al. (Mon,) studied this question.