Fluorimetric and high-performance liquid chromatographic determination of D-lactate in biological samples.
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 microliter.
Ohmori
S
S
Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Nose
Y
Y
Ogawa
H
H
Tsuyama
K
K
Hirota
T
T
Goto
H
H
Yano
Y
Y
Kondoh
Y
Y
Nakata
K
K
Tsuboi
S
S
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase; D007773:Lactates / Q000032:analysis* / Q000097:blood; D019344:Lactic Acid; D008099:Liver / Q000737:chemistry; D009243:NAD; D010655:Phenylenediamines; D011773:Pyruvates; D011786:Quality Control; D011810:Quinoxalines; D051381:Rats
D007202:Indicators and Reagents; D007773:Lactates; D010655:Phenylenediamines; D011773:Pyruvates; D011810:Quinoxalines; D009243:NAD; C047826:1,2-diamino-4,5-dimethoxybenzene; D019344:Lactic Acid; C070659:2-hydroxy-6,7-dimethoxy-3-methylquinoxaline; C044241:pyruvate hydrazone; D007770:L-Lactate Dehydrogenase