Determination of native folates in milk and other dairy products by high-performance liquid chromatography

Folates were measured in dairy products by high-performance liquid chromatography without prior sample clean-up. Detection limits for individual folates range from 0.3 to 7.3 ng/g. The folates were extracted from the sample matrix by adjusting the pH to 4.5 with acetic acid, centrifuging to remove precipitated proteins, and treating with conjugase to remove multiple polyglutamate residues. Folates were separated from other sample components using a reversed-phase column with a methanol-phosphate buffer (pH 6.8), and ion-pairing with tetrabutylammonium ion. Fluorescence was found to be the most useful detection technique. Fluorescence detection of reduced forms of the vitamin was achieved by post-column pH adjustment of the eluent with phosphoric acid, while the parent folic acid molecule required chemical oxidation with hypochlorite in order to obtain a fluorescent response.