Abstract
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.
Highlights
The L-enantiomer forms of amino acids greatly predominate in nature, D-enantiomers generated from the post-translational isomerization modifications on L-amino acids have been detected in bacteria, plants, in different phyla of invertebrates as well as in vertebrate animals up to mammals, including humans [1–5]
Many in vivo and in vitro studies have suggested that D-Asp has a crucial role in N-methyl D-Asp (NMDA) receptor-mediated neurotransmission playing very important functions in physiological and pathological processes [12–14, 31]
Limits of detection (LOD) obtained for derivatised amino acids analysed by different high-performance liquid chromatography (HPLC) methods resulted to be at least 10 times higher than the values obtained by Multiple Reaction Monitoring (MRM) measurements [35–37]
Summary
The L-enantiomer forms of amino acids greatly predominate in nature, D-enantiomers generated from the post-translational isomerization modifications on L-amino acids have been detected in bacteria, plants, in different phyla of invertebrates as well as in vertebrate animals up to mammals, including humans [1–5] Their appeal derives from the multiplicity of performed physiological functions, some of them not yet elucidated. In the brain, the strong decrease of free D-Asp levels after birth is a consequence of the postnatal onset of D-aspartate oxidase (DDO), the only known enzyme that catabolizes D-Asp [11]. In this organ, D-Asp appeared concentrated in the synaptic vesicles of terminal axon, suggesting its role as an endogenous neurotransmitter [10, 12]. A significant reduction of D-Asp levels has been recently found in the prefrontal cortex of patients with schizophrenia, associated with an increased expression of DDO mRNA [14]
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