Open AccessQuantitative determination of free D-Asp, L-Asp and N-methyl-D-aspartate in mouse brain tissues by chiral separation and Multiple Reaction Monitoring tandem mass spectrometry
Author(s) -
Carolina Fontanarosa,
Francesco Pane,
Nunzio Sepe,
Gabriella Pinto,
Marco Trifuoggi,
Marta Squillace,
Francesco Errico,
Alessandro Usiello,
Piero Pucci,
Angela Amoresano
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0179748
Subject(s) - chromatography , analyte , chemistry , repeatability , selected reaction monitoring , detection limit , tandem mass spectrometry , extraction (chemistry) , mass spectrometry , nmda receptor , liquid chromatography–mass spectrometry , receptor , biochemistry
Several studies have suggested that free d -Asp has a crucial role in N-methyl d -Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d -Asp, l -Asp and N-methyl d -Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l -Asp, d -Asp and N-methyl d -Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75–110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d -Asp, 0.46 pg/μl for l -Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d -Asp, 1.41 pg/μl for l -Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d -Asp, l -Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d -Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.