Determination of nicotinamide and metabolic products in urine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the quantitation of nicotinamide, nicotinic acid, nicotinuric acid, 1-methylnicotinamide and 1-methyl-2-pyridone-5-carboxamide in rat and human urines. The procedure utilizes a Varian Model 5020 liquid chromatograph with a UV detector, and an Altex 15 cm X 4.6 mm Ultrasphere-ODS column, employing a linear ion-pair mobile phase gradient. Solvent A contains 10 mM concentrations of pentanesulfonic acid (PSA), tetramethylammonium chloride (TMA) and KH2PO4, and solvent B contains PSA, TMA and acetonitrile. Different pH values for solvent A vary the retention times and thus the separation of the five compounds. Temperature of the system is critical. The conditions found most satisfactory were pH 3.30 and 24.5 degrees C.