A micellar electrokinetic chromatography-mass spectrometry approach using in-capillary diastereomeric derivatization for fully automatized chiral analysis of amino acids

For the first time, a fully automatized micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) method with in-capillary derivatization was developed for the chiral analysis of d- and l-amino acids using (−)-1-(9-fluorenyl) ethyl chloroformate (FLEC) as labeling reagent. The derivatization procedure was optimized using an experimental design approach leading to the following conditions: sample and FLEC plugs in a 2:1 ratio (15 s, 30 mbar: 7.5 s, 30 mbar) followed by 15 min of mixing using a voltage of 0.1 kV. The formed diastereomers were then separated using a background electrolyte (BGE) consisting of 150 mM ammonium perfluorooctanoate (APFO) (pH = 9.5) and detected by mass spectrometry (MS). Complete chiral resolution was obtained for 8 amino acids, while partial separation was achieved for 6 other amino acid pairs. The method showed good reproducibility and linearity in the low micromolar concentration range. The applicability of the method to biological samples was tested by analyzing artificial cerebrospinal fluid (aCSF) samples.