Abstract
We aimed to monitor changes in the levels of amino acid neurotransmitters or neuromodulators simultaneously at the synaptic clefts of experimental animals. We developed a method for the simultaneous determination of the levels of amino acids, such as d-Ser, Gly, and l-Glu, which were involved in neurotransmission via the N-methyl-d-aspartate (NMDA) receptor, and other protein-constituted amino acids in a rat brain microdialysis (MD) sample. We used a liquid chromatography-mass spectrometry (LC-MS)/MS device equipped with a triazole-bonded column. The determination was achieved without using stable isotope-labeled compounds. We instead used suitable amino acid analogues as internal standards (ISs). We examined various analyte-IS combinations to improve reproducibility. We found a positive correlation (r = 0.720, **p < 0.0001) between relative standard deviation (%) of the area ratio and the analyte-IS retention time differences. Using the proposed method, we were able to accurately analyze trace amounts of amino acids in MD samples using ISs that were structurally similar to the analytes. Furthermore, we observed that the peripheral administration of S-methyl-l-cysteine, which was an inhibitor of the amino acid transporter Asc-1, caused some amino acid level changes in the rat brain. The proposed LC-MS/MS method can be applied in vivo to study the effects of novel therapeutic agents with monitoring the levels of amino acid neuromodulators, such as Glu, Gly, GABA, and d-Ser, in the brain.
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Acknowledgements
The authors thank Mr. Tago for his technical assistance. The authors would also like to thank Shimadzu Access Corporation for the equipment conservation and technical advice. We also thank Editage (www.editage.jp) for the English language editing.
Sources of funding
This work was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C) (Grant Number 25460224), and the Faculty of Pharmaceutical Science, Toho University.
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The authors declare that they have no conflicts of interest.
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All of the animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals at Faculty of Pharmaceutical Sciences, Toho University. The project of animal experiments was approved by the Animal Care and Ethics Committee of Toho University (approval number: 16-55-165).
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Sakamoto, T., Onuma, R., Furukawa, S. et al. Liquid chromatography-mass spectrometry with triazole-bonded stationary phase for N-methyl-d-aspartate receptor-related amino acids: development and application in microdialysis studies. Anal Bioanal Chem 409, 7201–7210 (2017). https://doi.org/10.1007/s00216-017-0682-2
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DOI: https://doi.org/10.1007/s00216-017-0682-2