Chemoselective and enantioselective analysis of proteinogenic amino acids utilizing N-derivatization and 1-D enantioselective anion-exchange chromatography in combination with tandem mass spectrometric detection

d-Amino acid analysis in biological samples still poses a challenge to analytical chemists. In higher developed species trace amounts of d-amino acids have to be detected in vast excesses of the corresponding l-enantiomers. This method utilizes an easy-to-carry-out derivatization step on the amino group with an iron ferrocenyl propionate hydroxy succinimide ester followed by one-dimensional enantioselective anion exchange chromatography with cinchona alkaloid based chiral stationary phases (CSPs). MS detection is carried out in the highly sensitive SRM (selected reaction monitoring) mode, which allows a chemoselective differentiation of amino acid derivatives as well as their enantioselective separation in one step. Application of this method allows LOD (limits of detection) in the low μmol L−1 range and baseline enantioseparation for all proteinogenic amino acids except for Pro, Arg and His. The d-enantiomers of isomeric Leu and Ile were separated chromatographically and pose an example for the complementary selectivities of LC and MS. A successful application of this procedure to unprocessed human urine indicated the eligibility to analyse biological samples.

► SFP derivatization supports the enantiorecognition and increases chromatographic selectivity.
► SFP labelling enables selective and sensitive MS detection.
► SFT derivatization yields stable products.
► 19 chiral proteinogenic amino acids are enantioselectively and chemoselectively separated.