Evaluation of elution parameters in reversed phase HPLC for the determination of niacin vitamers and metabolites.

A procedure for separation of nicotinic acid (NIA), nicotinamide (NAM), nicotinuric acid (NUR) and N1-methyl-nicotinamide (NMN) from biological samples by isocratic HPLC is described. The effects a) of different buffer systems (citrate, acetate, propionate), b) of different pH values (between pH 2.4 and 3.35), c) of different ionic concentrations of the eluents (between 10 and 100 mM) and d) of different methanol concentrations added to the eluent on retention times of the four compounds were tested. Results were described by multiple regression equations which allow selection of suitable analytical conditions according to the specific composition of different biological materials. The procedure yielded satisfactory separation of NIA and NAM from various purified fractions of rumen and intestinal contents and blood.