Reversed-Phase Liquid Chromatographic Determination of Vitamin D in Infant Formulas and Enteral Nutritionals

Vitamin D In infant formulas and enteral nutritional products is determined by reversed-phase liquid chromatography (LC) with UV detection. The sample Is saponified 30 mln at 60°C and extracted Into 60 mL hexane. The hexane layer is then washed and evaporated to dryness. The sample Is reconstituted and added to a 3 mL silica solid-phase extraction column. Vitamins D2 and D3 are eluted from the column with 7 mL methylene chlorlde-lsopropanol mixture (99.8 + 0.2). The eluant is evaporated to dryness and reconstituted In 1 mLacetonltrlle. The acetonitrile solution is analyzed on a C18 reversed-phase LC column (25 cm x 4.6 mm, 5 μm particle size) with UV detection at 265 nm. Linearity for this method between 8 and 2600 lU/qt has shown a coefficient of determination of 1.000, with method precision ranging from 1 to 6%. Spike recoveries gave a mean of 99.1%. Because this method can quantitate and distinguish between vitamin D2 and vitamin D3 In products, vitamin D2 is used as an Internal standard In quantltatlng vitamin D3, and vice versa. The method Is applicable to milk-, soy-, and protein hydrolysate-based infant formulas and enteral nutritional products, both liquid and powder. The sample throughput is estimated to be 24 per day. An AOAC collaborative study of this method is recommended.