High-performance liquid-chromatographic determination of free nicotinic acid and its metabolite, nicotinuric acid, in plasma and urine.

We report a liquid-chromatographic procedure for determining free nicotinic acid and a metabolite, nicotinuric acid, in plasma and urine. Five-tenths milliliter of urine or deproteinized plasma is evaporated and the residue analyzed isocratically by reversed-phase ion-pair chromatography, with measurement of the eluted nicotinic acid and nicotinuric acid at 254 nm. Nicotinic acid, nicotinuric acid, and the internal standard (isonicotinic acid) have retention times of 7.8, 8.4, and 6.8 min, respectively, in plasma, and 12.3, 13.1, and 10.8 min in urine, because of double column length. Day-to-day reproducibilities (CV) for nicotinic acid and nicotinuric acid within 7.5% are attainable for the concentration ranges 0.1--20 mg/liter, equivalent to 0.81--162 micromol of nicotinic acid and 0.55--11 micromol of nicotinuric acid per liter for plasma; in urine for the range 0.5--100 mg/liter, equivalent to 4--810 micromol of nicotinic acid and 2.8--555 micromol of nicotinuric acid per liter. Metabolites of nicotinic acid such as nicotinamide, N-methylnicotinamide, 2-hydroxypyridine-5-carboxylic acid, and other structurally related substances do not interfere.