Liquid Chromatographic Determination of Vitamins D2 and D3 in Fortified Milk and Infant Formulas

Abstract

A liquid chromatographic (LC) method was developed for determining vitamins D2 and D3 in fortified milk and infant formulas. The lipid-soluble components were extracted from the aqueous phase by homogenizing in isopropanol-methylene chloride with magnesium sulfate added to remove water. The vitamins were fractionated from the lipid material by using gel permeation chromatography (GPC) followed by further cleanup of the combined GPC fractions on a muBondapak/NH2 column. Four muStyragel (100 A) columns connected in series were used for GPC fractionation of sample extracts in methylene chloride. Injection and collection were repeated 3 times to collect enough vitamin D for quantitation. The muBondapak/NH2 column, using a mobile phase of methylene chloride-isooctane-isopropanol (600 + 400 + 1), resolved vitamin D from other UV-absorbing compounds and soy sterols in infant formula and from cholesterol in milk. Vitamins D2 and D3 coeluted as one peak, with the resolution and vitamin level sufficient for visual monitoring (280 nm/0.02 absorbance unit full scale) in a collection time of 22-26 min. A Zorbax ODS (6 micron) column and a methylene chloride-acetonitrile-methanol (300 + 700 + 2) mobile phase were used for LC quantitation; vitamins D2 and D3 were baseline resolved in about 11 min. The infant formula samples included ready-to-use and concentrated liquids prepared in nonfat milk base or soy base fortified with vitamins D2 or D3 at 400 IU/qt or L (10 micrograms). The mean percent recovery of added vitamin D3 (400-500 IU/qt) from infant formula (n = 7) was 89.6 +/- 6.7 (coefficient of variation (CV) 7.5%).(ABSTRACT TRUNCATED AT 250 WORDS)