Quantification of D-amino acids in the central nervous system of Aplysia californica by liquid chromatography/tandem mass spectrometry

Abstract

A sensitive, specific and reliable liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for simultaneous determination of D-amino acids in the central nervous system (CNS) of Aplysia californica. In order to correct for any potential matrix effects on measured signals, deuterium-labeled L-Asp-d3 was used as an internal standard. Pre-column derivatization of the sample with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) allowed both effective in-line pre-concentration and sensitive MS/MS detection of the analytes. An extraction column (50x0.25 mm, 5 microm C18 silica particles) was used to pre-concentrate/stack samples. Enantiomeric separation of amino acid enantiomers was achieved on a chiral column packed with teicoplanin aglycone bonded silica particles (170x0.25 mm, 5 microm) with an MS-friendly mobile phase. The characteristic precursor to product ion transitions, m/z 297-->279 (for NBD-Asp), m/z 269-->223 (For NBD-Ser), m/z 311-->293 (for NBD-Glu) and m/z 300-->282 (for NBD-L-Asp-d3) were monitored for the quantification. Samples from the CNS of A. californica and heart tissues were analyzed. D-Asp was detected at high levels in all the ganglia and nerve tissues, but not in the heart tissue. Further, neither D-Ser nor D-Glu was detected in Aplysia, a widely used neuronal model.