Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 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 detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D-2, D-3, E, K-1, K-3) and several hydroxy metabolites of vitamin D-3 [25-(OH)-D-3, 24,25-(OH)(2)-D-3 and 1,25-(OH)(2)-D-3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration 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 vitamin A, K-1 and K-3 and between 1 and 100 ng ml(-1) for vitamin E, with excellent regression coefficients (greater than or equal to 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D-2, D-3, K-1 and K-3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D-3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.