Simultaneous Measurement of Amino Acid Enantiomers in Aged Mouse Brain Samples by LC/MS/MS Combined with Derivatization Using N α-(5-Fluoro-2,4-dinitrophenyl)-l-leucinamide (l-FDLA)

Abstract

D-amino acids have distinct roles from their l-enantiomer. In particular, some D-amino acids function as agonists or antagonists of neuronal receptors and are involved in higher brain functions. Thus, it is important to precisely measure the levels of these amino acid enantiomers in cells and tissues. Various quantification methods have been developed for measurements of chiral amino acids. However, each method has advantages and disadvantages. Additionally, measuring the amino acid enantiomers in crude biological samples requires a higher selectivity. In this study, we developed a quantification method for amino acid enantiomers using derivatization with N ^α-(5-Fluoro-2,4-dinitrophenyl)-l-leucinamide (l-FDLA) followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a conventional reversed-phase column. We simultaneously identified 10 chiral amino acids. Furthermore, we applied this method to investigate murine tissue samples and examined the effect of aging on the amino acid levels in aged brain regions. We found that aging decreased the levels of both D-serine and D-aspartate in the hippocampus. In addition, D-Phenylalanine in the thalamus significantly increased with age. In conclusion, our method is suitable for the quantification of the D-amino acids in crude biological samples and may contribute to elucidating the biological roles of chiral amino acids.

Keywords: D-amino acid; LC/MS/MS; aging; brain; l-FDLA.

Figure 1

Scheme of derivatization for amino acid enantiomers by N^α-(5-Fluoro-2,4-dinitrophenyl)-l-leucinamide (l-FDLA). l-FDLA-derivatized amino acids become diastereomeric and can be separated by a conventional reversed-phase high-performance liquid chromatography (HPLC) column.

Figure 2

Chromatograms of standard compounds of derivatized l- and d-amino acids. Standard l- and d-amino acids were derivatized with l-FDLA and analyzed by LC/MS/MS. Representative MRM chromatograms of derivatized l- and d-amino acids are presented as merged to confirm the drift of retention time. In each panel, relative ion abundance (MS counts) versus retention time (min) are shown.

Figure 3

Chromatograms of derivatized l-amino acids in the murine cortex samples. Tissue samples were extracted from the cortices of 3-month-old mice. Representative MRM chromatograms of derivatized l-amino acids are shown. In each panel, relative ion abundance (MS counts) versus retention time (min) are shown. Integrated MS count (green) represents the amount of target molecules.

Figure 4

Chromatograms of derivatized d-amino acids in the murine cortex samples. Tissue samples were extracted from the cortices of 3-month-old mice. Representative MRM chromatograms of derivatized d-amino acids are shown. In each panel, relative ion abundance (MS counts) versus retention time (min) are shown. Integrated MS count (green) represents the amount of target molecules.

Figure 5

Standard curves of derivatized l- and d-amino acids. Trends of MS count to amino acid concentrations were calculated by measuring the several standard solutions. The X-axis represents concentrations of amino acids before being derivatized, and the Y-axis represents the integrated sum of the peak area from each chromatogram. The equations and R² values of each plot are shown.