Purification procedure for the determination of niacin vitamers in gastrointestinal contents and blood.

A solid phase extraction procedure for subsequent simultaneous HPLC analysis of free nicotinic acid (NIA) and nicotinamide (NAM) in dried or liquid rumen and gastrointestinal contents from sheep is described. Twenty-five mg dried samples were suspended in 1.0 ml of 0.1 mol/l Li2SO4 and centrifuged. The supernatants were passed through two 500 mg solid phase extraction columns (Bond Elut NH2 and Sep Pak C18), mounted in series, and eluted by a four step pH gradient procedure. Recoveries of the two vitameres were between 70 and 75%. Purification was satisfactory when the method was applied to different fractions of rumen content (e.g. to food residues, to a microbial preparation and to rumen fluid), to intestinal contents of sheep and to blood. In rumen fluid no free NIA and NAM was found. Food particles and rumen microbes contained no free NAM.