Determination of niacin in infant formula by solid-phase extraction and anion-exchange liquid chromatography - PVM 1:2000

A peer-verified, solid-phase extraction (SPE)/anion exchange liquid chromat ographic method is presented for the determination of niacin in milk-based and soy-based infant formula. Analysis is in 3 steps: test sample digestion , extraction/cleanup, and liquid chromatography (LC). Digestion uses standa rd AOAC digestion procedure that involves autoclaving at 121 degreesC for 4 5 min in (1 + 1) H2SO4 to free endogenous niacin from protein and to conver t added niacinamide to niacin. The digest solution is adjusted to pH 6.5 wi th 7.5M NaOH. Acidification to pH <1.0 with (1 + 1) H2SO4 precipitates the protein. The clarified solution is then filtered, and the filtrate is broug ht to volume. SPE of niacin is accomplished by passing an aliquot of the di gest solution through an aromatic sulfonic acid-SPE (ArSCX-SPE) column. Aft er the column is washed with methanol and water to remove extraneous materi al, the niacin is eluted with 0.25M sodium acetate/acetic acid buffer at pH 5.6. An an- ion-exchange polystyrene-divinylbenzene column with 0. 1 M sod ium acetate/acetic acid buffer at pH 4.0 is used for LC. Niacin is determin ed by UV detection at 260 nm. A standard curve is prepared by passing known amounts of niacin through the ArSCX-SPE columns used for niacin extraction . The following values for x and relative standard deviation (RSD) were obt ained for National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1846 Infant Formula with a certified value for niacin of 63.3 +/- 7.6 mug/g: Submitting laboratory.-x = 59.7 +/- 4.0 mug/g RSD = >6.7%; confidence interval (CI) = +/- 1.4 mug/g; n = 27. Peer laboratory.-x = 56.6 +/- 6.6 mug/g; RSD = > 11.7%; CI = +/- 4.1 mug/g; n = 8.