During the course of establishing the advanced Marfey's method that has been developed to non-empirically determine the absolute configuration of constituent amino acids in a peptide using LC-MS, we encountered the "ornitine mystery" in the di-DLA (2,4-dinitrophenyl-5-leucinamide) derivative such that the elution order of ornitine (Orn) was opposite (D-->L) in spite of their relatively long retention time. In order to resolve this problem, the elution behavior of several mixed DLA and DPEA (2,4-dinitrophenyl-5-phenylethylamine) derivatives with different absolute configurations was carefully observed during HPLC. The length of the methylene chain in basic amino acids was obviously critical for this behavior, because Dab (2,4-diamino-n-butyric acid) and lysine (Lys) did not exhibit this abnormality. The presence of the carboxyamide moiety at the omega position was also essential for this phenomenon, because it was never observed in the DPEA derivatives at the omega position. Furthermore, it was found that the following combination of absolute configurations of Orn and DLA at the omega position only induced this abnormality: D-Orn and L-DLA, and L-Orn and D-DLA. This suggested that the structural interaction such as hydrogen bonding between the carboxyamide of DLA at the omega position and carboxylic acid at the alpha position in these derivatives reduced their retention power on the reversed-phase column.