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

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.

Fig 1. Total ion current (TIC) chromatogram for the analysis of 100 pg/μl standard mixture solution of l-Asp, d-Asp and NMDA.

Peaks corresponding to each analyte are labelled.

Fig 2. LCMSMS analyses in MRM mode of test samples from mouse brain tissues to optimise the sample treatment procedure.

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

Fig 3. MRM chromatogram for a DDO treated matrix sample only displaying the l-Asp component.

The specific MRM transitions for monitoring the l-Asp analyte are indicated.

Fig 4. Calibration curves obtained for the three analytes.

Fig 5. TIC and MRM chromatograms recorded for the three analytes in a sample from prefrontal cortex of wild type mouse.

The specific MRM transitions for each analyte are indicated.

Fig 6. 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.

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