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

Abstract

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, D₂, D₃, E, K₁, K₃) and several hydroxy metabolites of vitamin D₃ [25-(OH)-D₃, 24,25-(OH)₂-D₃ and 1,25-(OH)₂-D₃]. 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 D₂ and D₃ and their hydroxy metabolites, between 0.1 and 100 ng ml^–1 for vitamin A, K₁ and K₃ and between 1 and 100 ng ml^–1 for vitamin E, with excellent regression coefficients (≥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 D₂, D₃, K₁ and K₃ and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D₃ in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.