D-Amino acids (AAs) are increasingly being recognized as essential molecules in biological systems. Enantioselective analysis of proteinogenic AAs in biological samples was accomplished by CE-MS employing beta-CD as chiral selector and ESI via sheath-liquid (SL) interfacing. Prior to analysis, AAs were fully derivatized with FMOC, improving AA-enantiomer separation and ESI efficiency. In order to optimize the separation and MS detection of FMOC-AAs, the effects of type and concentration of CD in the BGE, the composition of the SL, and MS-interfacing parameters were evaluated. Using a BGE of 10 mM beta-CD in 50 mM ammonium bicarbonate (pH 8) containing 15% v/v isopropanol, a SL of isopropanol-water-1 M ammonium bicarbonate (50: 50: 1, v/v/v) at a flow rate of 3 mu L/min, and a nebulizer gas pressure of 2 psi, 15 proteinogenic AAs could be detected with enantioresolutions up to 3.5 and detection limits down to 0.9 mu M (equivalent to less than 3 pg AA injected). The selectivity of the method was demonstrated by the analysis of spiked cerebrospinal fluid, allowing specific detection of D-AAs. Repeatability and linearity obtained for cerebrospinal fluid were similar to standard solutions, with peak area and migration-time RSDs (n = 5) below 16.2 and 1.6%, respectively, and a linear response (R-2 >= 0.977) in the 3-90 mu M range.