Determination of vitamins D-2, D-3, K-1 and K-3 and some hydroxy metabolites of vitamin D-3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography UV detection

A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV dete ction is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic det ection system. The overall process was developed and applied to the main li posoluble vitamins (A, D-2, D-3, E, K-1, K-3) and several hydroxy metabolit es of vitamin D-3 [25-(OH)-D-3, 24,25-(OH)(2)-D-3 and 1,25-(OH)(2)-D-3]. Al l the analytes were monitored at a compromise wavelength of 270 nm. Calibra tion graphs were constructed between 0.01 and 100 ng ml(-1) for vitamin D-2 and D-3 and their hydroxy metabolites, between 0.1 and 100 ng ml(-1) for v itamin A, K-1 and K-3 and between 1 and 100 ng ml(-1) for vitamin E, with e xcellent regression coefficients (greater than or equal to 0.9901) in all c ases. The precision was established at two concentration levels with accept able RSDs in all instances (between 3.6 and 8.7%). The method was appropria te for the determination of vitamin D-2, D-3, K-1 and K-3 and the 24,25-dih ydroxy and 25-hydroxy metabolites of vitamin D-3 in human plasma. The metho d was applied to plasma samples spiked with the target analytes and the rec overies ranged between 78 and 109%.