Calibration curves obtained for the three analytes.

<p>Calibration curves obtained for the three analytes.</p

Quantitative determination of l-Asp, d-Asp and NMDA in samples from mouse prefrontal cortex by MRM LC-MSMS analysis.

<p>Samples were obtained from brain of three individual animals from each group (<i>DDO</i>^+/+, wild type mouse; <i>DDO</i>^-/-, D-aspartate oxidase (DDO) knockout mouse). Each measurement represents the average of three technical replicates.</p

TIC and MRM chromatograms recorded for the three analytes in a sample from prefrontal cortex of wild type mouse.

<p>The specific MRM transitions for each analyte are indicated.</p

Quantitative 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

<div><p>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.</p></div

Total ion current (TIC) chromatogram for the analysis of 100 pg/μl standard mixture solution of l-Asp, d-Asp and NMDA.

<p>Peaks corresponding to each analyte are labelled.</p

Quantitative analysis of d-Asp (left panel) and d-Asp versus l-Asp ratio (right panel) in samples from mouse prefrontal cortex using the developed LC-MS/MS MRM procedure.

<p><i>DDO</i>^+/+, wild type mouse; <i>DDO</i>^-/-, d-aspartate oxidase (DDO) knockout mouse.</p

LCMSMS analyses in MRM mode of test samples from mouse brain tissues to optimise the sample treatment procedure.

<p>Some experimental conditions for sample extraction and precipitation are reported in the figure. Peaks corresponding to each analyte are labelled.</p

Validation parameters of the analytical method developed for the quantitative determination of l-Asp, d-Asp and NMDA.

<p>Each measurement represents the average of three technical replicates.</p