Determination of water-soluble vitamins in infant milk by high-performance liquid chromatography

A rapid, simple and reliable liquid chromatographic method has been developed for the simultaneous determination of nicotinamide, thiamin, riboflavin, pyridoxine, pyridoxal, pyridoxamine, cyanocobalamine and folic acid in liquid and powdered infant milk. Ion-pair chromatography with a reversed-phase C18 column is used. Six vitamins were resolved in a single analysis; total analysis time never exceeded 55 min. A mobile phase of methanol-water (15:85), 5 mM octanesulfonic acid, with 0.5% triethylamine at pH 3.6 and a flow-rate of 1.0 ml/min gave the most satisfactory separation of these vitamins using a UV detector set at different wavelengths. Sample preparation involves acidification to precipitate proteins, and centrifugation followed by gravity filtration. Linearity, precision, recovery and sensitivity were always satisfactory. Detection limits ranged from 0.02 to 0.10 microgram/ml and determination limits ranged from 0.03 to 0.25 microgram/ml.