Enzymatic cycling assay for D-carnitine determination

An enzymatic method for D-carnitine determination using the enzyme D-carnit ine dehydrogenase is described. The assay is based on the amplified signal produced during NAD(+) cycling in the presence of a tetrazolium salt and us ing phenazine methosulfate as electron carrier. Optimum assay conditions we re studied with two tetrazolium salt pairs: 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)/MTT-formazan and 2-(4-iodophenyl)-3-(4 -nitrophenyl)-5-phenyl tetrazolium chloride (INT)/INT-formazan. The first p air (MTT) showed higher sensitivity. The calibration curve was linear from 0.1 to 5 mM D-carnitine, with a quantification limit of 0.1 mM and a relati ve standard deviation of 1.51%. The procedure is simple, rapid, accurate, a nd easily automated. It was satisfactorily applied to following D-carnitine levels during the microbial transformation of D-carnitine into L-carnitine and to determining the D-carnitine content of pharmaceutical preparations. (C) 1999 Academic Press.