Chiral analysis of amino acid neurotransmitters and neuromodulators in mouse brain by CE‐LIF

Abstract

Chiral CE method has been developed for quantitative determination of d-amino acid modulators of NMDA glutamate receptor; d-serine and d-aspartate along with l-glutamate and l-aspartate in biological samples. These ligands are suggested to be involved in regulation of NMDA receptor related brain functions, such as neurogenesis, neuronal plasticity, and memory formation. For sensitive determination of the amino acids LIF detection was chosen, and a fluorogenic reagent, 7-fluoro-4-nitro-2,1,3-benzoxadiazole was used for derivatization. An amino-modified β-CD, 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-CD (HPA-β-CD) was applied as chiral selector. Determinations were accomplished in a polyacrylamide coated capillary and reverse polarity was used for the analysis of the negatively charged analytes. The method was optimized and validated; 6 mM HPA-β-CD in 50 mM HEPES buffer, pH 7 was appropriate to achieve baseline separation of the analytes. The limit of quantification with acceptable accuracy is 0.05 μM for both d-amino acids. The method was used for the determination of d-aspartate and d-serine content in various brain regions of adult mice.

Improving the sensitivity in chiral capillary electrophoresis

“…LIF is the detection system which offers the best sensitivity of all the detectors used in CE and an evidence of this is the low LODs depicted in Table , generally in the nanomolar range even though the reported works did not employ additional preconcentration strategies . Moreover, in a recent published review article, it was demonstrated that LIF has been the most employed detector system in CE to determine chiral neuroactive molecules (e.g.…”

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

“…CE laser induced fluorescence (LIF) detection systems are increasingly used for the chiral separation of all the amino acids due to the high separation efficiency, and compatibility with sensitive detection methods including MS instruments [43]. Although the LOD by such method achieves concentration of the low nM for several molecules [44], recent data related to d -Asp quantification showed LOD of 17 nM (2.3 ppb) in chicks brain tissues [45] and 8 nM (1 ppb) in various brain regions of adult mice [46], 4 and 2 fold, respectively, higher than LOD recorded with our MRM method. Peak assignments for most of these methods needed to be confirmed [47].…”

Recent development of cationic cyclodextrins for chiral separation

“…The sensitive determination of amino-acid neurotransmitters and neuromodulators in mouse brain was achieved by CE-LIF using 7-fluoro-4-nitro-2,1,3-benzoxadiazole for derivatization and HPrAMCD as chiral selector [35]. The limit of quantification (LOQ) was obtained an accuracy of 0.05 µM for D-serine and D-aspartate.…”