HPLC analysis of quinolinic acid, a NAD biosynthesis intermediate, after fluorescence derivatization in an aqueous matrix.
Quinolinic acid (2,3-pyridine dicarboxylic acid), a biological intermediate in nicotinamide adenine dinucleotide (NAD) biosynthesis in microbes and mammals and a brain excitotoxin, is not fluorescent nor electrochemically active and its detection sensitivity by UV absorption is comparatively low. Quinolinic acid was successfully derivatized in water-based samples by monodansylcadaverine, a fluorescence tag, and analysed by high-performance liquid chromatography (HPLC). No extraction procedure was needed and quinolinic acid was activated by water-soluble carbodiimide and derivatized under mild conditions. As little as 3 pmol (500 pg) of quinolinic acid in 5 microliter of artificial cerebrospinal fluid sample volume could be derivatized and detected at a signal to noise ratio of 3:1. Thus, detection on a mass basis by HPLC after fluorescence derivatization is about 300 times as sensitive as direct determination of quinolinic acid by UV absorbance (500 pg vs 150 ng). A variety of activators, fluorescent tags and reaction solvents and conditions were tested but found to be less effective.
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Dalton Cardiovascular Research Center, University of Missouri-Columbia 65211, USA.
Dang
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Brown
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D002103:Cadaverine / Q000031:analogs & derivatives / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009243:NAD / Q000096:biosynthesis; D017378:Quinolinic Acid / Q000032:analysis* / Q000134:cerebrospinal fluid; D012680:Sensitivity and Specificity; D014867:Water
D005456:Fluorescent Dyes; D014867:Water; D009243:NAD; D017378:Quinolinic Acid; C008542:monodansylcadaverine; D002103:Cadaverine