RAPID ASSAY FOR THE DETERMINATION OF 2 PHOTOACTIVATIBLE KAINIC ACID ANALOGS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

An anion-exchange isocratic high-performance liquid chromatographic me thod for the separation and determination of two kainic acid analogues , namely -(4'-azidobenzoyl)aminomethylethenyl]-2-carboxy-3- pyrrolidin eacetic acid and enyl)thioureylenemethylethenyl]-2-carboxy-3-pyrro- li dineacetic acid is described. These kainic acid analogues were obtaine d by coupling dimethyl (2S,3S,4S)4-( 1-(9-fluorenylmethoxycarbonyl)-3- pyrrolidineacetic acid with N-hydroxysuccinimidyl 4-azidobenzoate and 4-azidophenyl isothiocyanate respectively, followed by saponification. The thus obtained products were purified by reversed phase flash colu mn chromatography and separated on Econosphere -NH2, using 5% (v/v) ac etonitrile in 50 mM acetate buffer PH 5.5, at a flow rate of 1.5 ml/mi n. The effect of mobile phase pH, and of the acetonitrile concentratio n on the resolution between kainic acid and its analogues was also stu died. Kainic acid was determined by using either low ultraviolet detec tion (190 nm) or the ninhydrin reaction. Detection of the eluted kaini c acid analogues was performed at 215 nm. The detection limit is appro ximately 1.5 pmol for both kainic acid analogues and the calibration g raphs were linear up to 100 nmol(approximately 50-mu g).