Determination of vitamin D3 content in cod liver oil using column-switching technique.

Objectives: To develop a two-dimensional liquid chromatography method to determine the content of vitamin D₃ in cod liver oil preparations.

Methods: The samples were prepared by saponification and extraction, and the content of vitamin D₃ was determined by two-dimensional liquid chromatography with dual-pump-single-valve switching dual detectors. The chromatographic column, capture device, and detection wavelength were optimized; the linearity, system suitability, recovery rate, repeatability and sample stability of the method were investigated, and further validated in actual sample determination.

Results: A column switching two-dimensional chromatography method was developed. In the first dimensional chromatography, an Agilent PoroShell SB-C8 (50 mm×4.6 mm, 2.7 μm) column was used, and acetonitrile-water was used as mobile phase and gradiently eluted at a flow rate of 1.0 mL/min, the detection wavelength was 264 nm. An Agilent Zorbax SB-C18 (150 mm×3.0 mm,1.8 μm) column was used in the second dimensional chromatography, and acetonitrile was used as mobile phase at a flow rate of 0.42 mL/min, the detection wavelength was 264 nm. In determining content of Vitamin D₃, there was a good linear relationship in the concentration range and the system suitability, recovery rate, repeatability and sample stability all met the verification requirements.

Conclusions: The developed two-dimensional liquid chromatography method in the study is accurate, reproducible and simple, it can simultaneously separate pre-vitamin D₃, trans-vitamin D₃, vitamin D₃, and tachysterol3, the baseline separation was achieved, indicating that it is worth promoting for application.