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Postcolumn Fluorimetric HPLC Procedure for Determination of Niacin Content of Cereals¹

¹Journal paper 3051 of the South Dakota Agricultural Experiment Station. Published with the approval of the director of the South Dakota Agricultural Experiment Station. This study was supported by the South Dakota Agricultural Experiment Station, South Dakota State University, and the South Dakota Wheat Commission. ²Associate professor and professor, respectively, Department of Nutrition and Food Science, South Dakota State University, Brookings, SD 57007-0497. ³Corresponding author. E-mail: KrishnP@ur.sdstate.edu Phone: 605/688-4040. Fax: 605/688-5603. ⁴Scientist 2, Novartis Pharmaceuticals Corporation, Lab 3NW, New Building, 25 Old Mill Road, Suffern, NY 10901.

Analytical procedures for determining niacin or vitamin B3 content of foods are tedious, require large quantities of toxic chemicals, and are timeconsuming. In addition, food matrices are difficult as samples because of their complex nature. A selective, sensitive HPLC technique was developed with postcolumn derivatization as well as fluorescence and spectroscopic detection systems. Niacin was separated and retained for 6.5 min on a polymeric column with an aqueous mobile phase containing sodium acetate buffer. A postcolumn system consisting of a stainless-steel pump and reaction coil allowed detection and quantitation of niacin. An acid-enzyme sample-extraction method was most compatible with HPLC and postcolumn derivatization with 5% each of acidified p-aminophenol and cyanogen bromide. Lower detection limit and mean recovery were 3.6 ng and 99.43%, respectively. Fluorescence response for nicotinamide was half that of nicotinic acid. A lower response for nicotinamide was also noted with conventional spectroscopy. However, the new method yielded comparable values for six of eight ready-to-eat commercial cereal samples. No significant difference was observed between the AACC reference and HPLC fluorimetric methods. Chemical derivatization was done within a reaction coil with reagents at half strength, limiting exposure to hazards and minimizing waste-disposal problems.