Capillary electrophoresis-mass spectrometry of derivatized amino acids for targeted neurometabolomics - pH mediated reversal of diastereomer migration order.

A targeted CE-MS approach was developed for the chiral analysis of biologically relevant amino acids in artificial cerebrospinal fluid (aCSF). In order to achieve chiral resolution, the five amino acids (Ser, Asn, Asp, Gln and Glu) were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate ((+)-FLEC). The diastereoselectivity was found to be highly dependent on pH for all analytes and the optimized background electrolyte (BGE) consisted of 150 mM acetic acid, adjusted to pH 3.7 with NHOH. Furthermore, a reversal of the migration order of Asp derivatives was observed. This phenomenon seems to be caused by intra-molecular interactions affecting the pK of the second ionizable group (the side chain carboxyl). The applicability of this method was evaluated using aCSF. A solid phase extraction (SPE) protocol was developed for the selective extraction of the FLEC derivatives. A full evaluation of the matrix effect and extraction yield was performed concluding that the matrix effect is marginal and the recoveries are between 46 and 92%. The method offers adequate sensitivity (limits of detection below 1 μM).