Selectivity evaluation of phenyl based stationary phases for the analysis of amino acid diastereomers by liquid chromatography coupled with mass spectrometry.

D-amino acids (AA) analysis is becoming more and more relevant for metabolomics, therefore new analytical tools need to be developed. A common approach to achieve AA enantioseparation is chiral derivatization. Among the chiral derivatization reagents, (+) or (-)-1-(9-fluorenyl) ethyl chloroformate ((+) or (-)-FLEC) has proved to be one of the most versatile. Suitable chiral selectivity for FLEC derivatives of amino acids could be obtained in reversed-phase HPLC using nonpolar stationary phases (C4, C8 and C18) and tetrahydrofuran (THF) based mobile phases. This study is meant to provide alternatives to the use of THF as organic modifier by evaluating the selectivity obtained on two phenyl based stationary phases for 19 FLEC-DL-AA pairs of diastereomers using UHPLC-MS. Several mobile phases consisting of ammonium acetate and different common organic solvents (acetonitrile (ACN), methanol (MeOH), 2-propanol (IPA)) were tested using gradient elution. Experimental design was employed for the optimization of the separation conditions. In the optimized conditions, complete chiral separation can be achieved for 18 out of 19 FLEC-DL-AAs in less than 30 min.