d-Lactate in biological samples was converted into a strongly fluorescent substance in a one-vial reaction. It was first converted into the pyruvate hydrazone in the presence of d-lactate dehydrogenase, an NADH-reoxidation system using diaphorase, d,l-6,8-thioctamide and hydrazine. This hydrazone was then converted into 2-hydroxy-6,7-dimethoxy-3-methylquinoxaline by 1,2-diamino-4,5-dimethoxybenzene in 1 M hydrochloric acid, and the quinoxaline was extracted and measured fluorimetrically at 432 nm (excitation at 365 nm). The calibration curve for d-lactate was linear up to at least 100 nmol/ml of the assay mixture, with a determination limit of 2 nmol/ml. The quinoxaline was also analysed by high-performance liquid chromatography with fluorimetric detection. The calibration curve for d-lactate was linear from 500 fmol to 75 nmol in the reaction mixture. This method was 4000 times more sensitive than the fluorimetric method, and could determine d-lactate in blood plasma volumes of less than 1 μl.
|Number of pages||8|
|Journal||Journal of Chromatography B: Biomedical Sciences and Applications|
|Publication status||Published - May 3 1991|
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