TY - JOUR
T1 - Fluorimetric and high-performance liquid chromatographic determination of d-lactate in biological samples
AU - Ohmori, Shinji
AU - Nose, Yuko
AU - Ogawa, Hiroko
AU - Tsuyama, Kohichi
AU - Hirota, Takashi
AU - Goto, Hiroshi
AU - Yano, Yoshiko
AU - Kondoh, Yasunori
AU - Nakata, Kunihiko
AU - Tsuboi, Seiji
N1 - Funding Information:
This work was supported in part by a Grant-in-Aid for Scientific Research (No. 62570986f)r om the Ministry of Education, Sciencea nd Culture, Japan.
PY - 1991/5/3
Y1 - 1991/5/3
N2 - 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.
AB - 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.
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U2 - 10.1016/0378-4347(91)80105-L
DO - 10.1016/0378-4347(91)80105-L
M3 - Article
C2 - 1885704
AN - SCOPUS:0025810557
SN - 0378-4347
VL - 566
SP - 1
EP - 8
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
IS - 1
ER -