Elution behavior of diaminopimelic acid and related diamino acids using the advanced Marfey's method

The advanced Marfey's method consists of a chromatography technique for the separation of amino acids into each enantiomer by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA), and a detection method using liquid chromatography/mass spectrometry (LC/MS) which can determine the non-empirically the absolute configuration of various amino acids including the non-protein ones. However, this method has not been applied to the determination of the absolute configuration of an amino acid with a "meso" configuration such as diaminopimelic acid (A2pm). In the present study, this method was successfully applied to determine the absolute configurations of diaminosuccinic acid (DAS), A2pm, cystine (Cys), selenocystine (SeCys) and homocystine (HomoCys) using a racemization procedure and the DL-FDLA method, and the resulting elution behavior was summarized as follows: (1) the LL- and meso-isomers were eluted prior to the DD-isomer except for one case; (2) the LL- and meso-isomers are closely eluted and the elution was occasionally reversed; (3) the retention time for both the L- and D-derivatives of the meso-isomer was not changed; (4) the complementary use of the two solvent systems using CH3CN and MeOH was effective to obtain a chromatogram with a high resolution; (5) the abnormality, such as the elution order and peak shape, was observed in the elution behavior of DAS.