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Determination of N-methyl-d-aspartate in tissues of bivalves by high-performance liquid chromatography
Institution:1. Laboratory of Neurobiology, Instituto de Investigación Médica Mercedes y Martin Ferreyra, INIMEC-CONICET-Universidad Nacional de Córdoba, Argentina;2. Facultad de Psicología, Universidad Nacional de Córdoba, Argentina;3. Department of Neurology, University of Texas Medical School at Houston, Houston, USA;1. Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States;2. Psychogenics Inc 215 College Road Paramus, NJ 07652, United States;3. Department of Pathology, Columbia University College of Physicians & Surgeons, New York, NY 10032, United States;4. HumanFirst Therapeutics LLC, Silver Spring, MD 20910, United States
Abstract:The natural occurrence of N-methyl-d-aspartate (NMDA) is limited to the foot muscle of Scapharca broughtonii; it is a well known compound for its neuroexitatory activity. This paper describes a high-performance liquid chromatographic (HPLC) method for the determination of NMDA in biological extracts. The method involves removal of neutral and basic substances by anion-exchange chromatography and removal of acidic primary amino acids by treatment with o-phthalaldehyde before derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate, followed by HPLC with isocratic elution with a selected mobile phase that separates the two diastereomers formed. The identity of the detected NMDA has been confirmed by a procedure using (−)-1-(9-fluorenyl)ethyl chloroformate as a derivatizing agent. The identification has been further supported by the disappearance of the peak of the NMDA derivative by pretreatment of the sample with d-aspartate oxidase. Application of the method has shown the presence of NMDA in several tissues of S. broughtonii and Scapharca subcrenata.
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