Modifications of AOAC Official Method 999.15 to Improve the Quantitation of Vitamin K1 in Complex Formulated Nutritional Products

Vitamin K-1 (phylloquinone) occurs in foods in relatively low concentrations. It is synthesized for addition to formulated nutritional products (infant formulas, medical foods, and adult nutritional products). In recent years, nutritional products formulated with free amino acids and partially hydrolyzed proteins have been introduced in the market. Schimpf et al. demonstrated that the current AOAC Official Method 999.15 for determination of vitamin K in milk and infant formula is not adequate to quantitatively extract vitamin K-1 from such products. We developed a modification of AOAC 999.15 for the analysis of vitamin K-1 in these products that provides quantitative extraction by increasing the sample size, volume of extraction solvents, time of liquid/liquid partitioning, and order of the addition of solvents. This modified procedure showed extraction efficiency comparable to that of the original AOAC 999.15 procedure for analyzing infant formula matrixes and to the modified procedure of Schimpf et al. for the analysis of samples containing limited amounts of free amino acids and/or partially hydrolyzed proteins. Extraction efficiency increased more than 10% using the modified extraction procedure for samples containing higher amounts of these components. The chromatographic separation was improved by using a Dionex Acclaim triacontanol-bonded C30 column (250x3.0 mm id, 3 mu m particle size) maintained at 15 degrees C, with acetonitrile methanol (50+50, v/v) mobile phase at a flow rate of 0.5 mL/min, which provided baseline separation of the cis and trans isomers of vitamin K-1 from each other and from other compounds contained in the sample extracts.