Journal Article

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

Squillace, Marta, Usiello, Alessandro, Amoresano, Angela, Pane, Francesca, Trifuoggi, Marco, Pinto, Gabriella, Pucci, Piero, Fontanarosa, Carolina, Sepe, Nunzio, Errico, Francesco

2017

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Amino acids / Analysis / Analytical chemistry / Animal tissues / Animals / Aspartic Acid - metabolism / Biology and Life Sciences / Biotechnology / Brain / Brain - metabolism / Buffers / Calibration / Capillary electrophoresis / Chromatography / Chromatography, Liquid / Enzymes / Extraction / Glutamate receptors / Glutamic acid receptors / Growth hormones / Ions / Limit of Detection / Linearity / Mammals / Mass spectrometry / Mass spectroscopy / Medicine and Health Sciences / Metabolites / Mice / Monitoring / N-methyl-D-aspartate / N-Methyl-D-aspartic acid / N-Methylaspartate - metabolism / Neurotransmission / Physical Sciences / Physiological aspects / Physiology / Reference Standards / Reproducibility / Reproducibility of Results / Research and Analysis Methods / Scientific imaging / Separation / Solvents / Stereoisomerism / Tandem Mass Spectrometry - methods / Tissues / Water analysis