Determination of N-methyl-D-aspartate in tissues of bivalves by high-performance liquid chromatography

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.