Reversed-phase high-performance liquid chromatography of nicotinic acid mononucleotide for measurement of quinolinate phosphoribosyltransferase.
A system has been developed for the determination of quinolinate phosphoribosyltransferase (QPRT) activity in liver and kidney homogenates using HPLC. A product, nicotinic acid mononucleotide (NaMN), is separated by reversed-phase chromatography (a Tosoh ODS 80TS was used as an analytical column) using a mixture of 10 mM KH2PO4-K2HPO4 buffer (pH 7.0) containing 1.48 g/l tetra-n-butylammonium bromide-acetonitrile (9:1, v/v) as a mobile phase. The flow-rate was 1.0 ml/min, the detection wavelength was 265 nm. The column temperature was maintained at 40 degrees C. Under these conditions, NaMN was eluted at about 8.1 min. Sample preparation was very straightforward. The reaction mixture of QPRT assay was stopped by immersing the tube into a boiling water bath, the resulting supernatant was filtered, and the filtrate was directly injected into a HPLC system. The total HPLC analysis time was approximately 20 min.
Shibata
K
K
Department of Life Style Studies, School of Human Cultures, The University of Shiga Prefecture, Hikone, Japan. kshibata@shc.usp.ac.jp
Fukuwatari
T
T
Sugimoto
E
E
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008297:Male; D009537:Nicotinamide Mononucleotide / Q000031:analogs & derivatives* / Q000032:analysis*; D010430:Pentosyltransferases / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)
D009537:Nicotinamide Mononucleotide; C002953:nicotinate mononucleotide; D010430:Pentosyltransferases; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)