Introduction

Vitamin, included in five nutritious  components,  is an essential to get oneself into good shape. Fat-soluble vitamin, which is in biological body of plants and animals, especially influences physiological activities of metabolism. The fat-soluble vitamin  has some characteristic features in terms of solubility and stability. In solubility, the fat-soluble vitamins has poor water solubility but has rich oil solubility. And, in stability, the fat-soluble vitamins is easily decomposed  by light and oxygen. These features make difficult the analysis of fat-soluble vitamins. HPLC is an effective tool to analyze these samples because it has capability to control the various features in measurement conditions.

In this article, the analysis of 8 components of fat-soluble vitamin is implemented.

LC-4000 HPLC system

Experimental

Experimental Condition
Column: Shodex Asahipak ODP-50G 4A (4.6 mmI.D. x 10 mmL, 5 µm) + Shodex Asahipak ODP-50 4E (4.6 mmI.D. x 250 mmL, 5 µm)
Eluent : Acetonitrile/Methanol (50/50)
Flow rate: 0.6 mL/min
Column temp.: 30 ºC
Injection volume: 20 µL
Standard: 8 fat-soluble vitamins

Keywords

Fat-soluble vitamin, Vitamin K3, Vitamin A, Retinol acetate, Vitamin D2, Vitamin D3, α-tocopherol, α-tocopherol acetate, Vitamin K1, Shodex Asahipak ODS-50 4E, PDA detector

Results

Figure 1 shows the chromatograms and contour map of the samples. The eight components are separated clearly in 25 min.

Figure 1. Chromatogram of fat-soluble vitamins (1: Vitamin K3 (Menadione) 14 µg/mL, 2: Vitamin A (Retinol)15 µg/mL, 3: Vitamin A acetate (Retinol acetate) 36 µg/mL, 4: Vitamin D2 (Ergocalciferol) 20 µg/mL, 5: Vitamin D3 (Cholecalciferol) 14 µg/mL, 6: Vitamin E acetate (α-Tocopherol acetate) 99 µg/mL, 7: Vitamin E (α-Tocopherol) 103 µg/mL, 8: Vitamin K1 (Phytonadione) 13 µg/mL)

Figure 2 shows the on-peak spectra of samples. As shown in below figures, fine spectra are obtained.

Figure 2. On-peak spectrum (top) and chromatogram (bottom) of Fat-soluble vitamins (1: Vitamin K3 (Menadione) 14 µg/mL, 2: Vitamin A (Retinol)15 µg/mL, 3: Vitamin A acetate (Retinol acetate) 36 µg/mL, 4: Vitamin D2 (Ergocalciferol) 20 µg/mL, 5: Vitamin D3 (Cholecalciferol) 14 µg/mL, 6: Vitamin E acetate (α-Tocopherol acetate) 99 µg/mL, 7: Vitamin E (α-Tocopherol) 103 µg/mL, 8: Vitamin K1 (Phytonadione) 13 µg/mL)