Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography.

In the present work, a reversed-phase high-performance liquid chromatographic procedure has been developed for the determination of water-soluble vitamins (thiamine hydrochloride, pyridoxine hydrochloride, nicotinamide, riboflavin phosphoric ester and cyanocobalamine) and fat-soluble vitamins (retinol palmitate, cholecalciferol, alpha-tocopherol acetate) in multi-vitamin pharmaceutical formulations. The sample treatment proposed consists of a solid-phase extraction with C18 AR cartridges that allow the separation of fat-soluble vitamins, which were retained on the sorbent, from water-soluble vitamins. Afterwards, the water-soluble vitamins were analysed by HPLC on a Nova-Pack C18 (150x3.9 mm, 4 microm) analytical column, using CH3OH-0.05 M CH3COONH4 as mobile phase. The chromatographic analysis of the fat-soluble vitamins was carried out after their sequential elution with methanol and chloroform from C18 sorbent, on the above column. The mobile phase employed was MeOH-CH2CN (95:5, v/v) working at a flow-rate of 2 ml min(-1) in isocratic mode. The solid-phase extraction for these vitamins had been previously optimised. The experimental variables studied were: application volume, elution solvents and cleaning solutions. The UV-Vis detection of vitamins was made at 270 nm for all the water-soluble vitamins (362 nm for B12) and 285 nm for the water-soluble and fat-soluble vitamins present in real samples at different concentration levels. The accuracy of the method was tested obtaining an average recovery ranging between 78 and 116%.