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

摘要

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-d(3) 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 (50 X 0.25 mm, 5 mu M C-18 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 (170 X 0.25 mm, 5 mu m) 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-d(3)) 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. Copyright (c) 2006 John Wiley & Sons, Ltd.