Analysis of Fat-Soluble Vitamins by HPLC

Udo Huber Pharmaceutical

Vitamins are compounds, which are necessary to maintain a healthy and properly functioning human organism. A few milligrams a day are enough to regulate the utilization of the nutrients, such as carbohydrates, fats, proteins and minerals. Usually vitamins are supplied in food. Figure 1 shows the separation of the three fat-soluble vitamins retinol (A), cholecalciferol (D3) and a-tocopherol (E) using gradient analysis on a reversed phase column and UV detection. The autosampler temperature was set to 4 ºC to avoid decomposition of the samples.

Conditions
Absorbance [mAU] 800 600 400 200 0 0 2.5 5 7.5 10 12.5 15 Time [min] 17.5 20 1 2 1 Retinol (A) 2 Cholecalciferol (D3) 3 α-Tocopherol (E) 3 Column 4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 µm Mobile phase A = water, B = methanol Flow rate 1.0 ml/min Gradient at 0 min 90 % B at 15 min 100 % B at 20 min 100 % B Column wash at 21 min 90 % B UV detector variable wavelength detector 210 nm, standard cell Column compartment temperature 20 °C Stop time 21 min Post time 5 min Injection volume 5 µl

Figure 1 Analysis of three fat-soluble vitamins

Agilent Technologies
Innovating the HP Way

04 Precision of Area (RSD of 10 runs) (1000 mg/l)* 0. 5 µm. Agilent Technologies Innovating the HP Way . The values for LOD. the Agilent 1100 Series thermostatted autosampler. 10/pk) Note: Since the method was specifically developed on the Agilent 1100 Series system you might not be able to reproduce this analysis on an older system or even on a new system with lower performance. standard flow cell 10-mm path length. cholecalciferol (D3) and a-tocopherol (E). 4 x 4 mm (Agilent part number 7995118-504. 13-µl cell volume • Agilent ChemStation + 3D software Columns • Zorbax Eclipse XDB.0 2. 13-µl cell volume Alternative: • Binary pump • Vacuum degasser • Diode array detector standard flow cell 10-mm path length. 4. precision of RT and precision of area show the good performance of the analysis. 3. 5 µm.10 0.04 0.0 Precision of RT (RSD of 10 runs) (1000 mg/l)* 0.6 x 75 mm (Agilent part number 7995118-344) • Recommended: Guard cartridges Zorbax Eclipse XDB-C18.HPLC Performance Compound LOD for S/N=2 (mg/l)* 4.20 Equipment Agilent 1100 Series • Quaternary pump (includes vacuum degasser) • Thermostatted autosampler • Thermostatted column compartment • Variable wavelength detector.5 2. To avoid sample decomposition it is necessary to use a cooled autosampler.16 0. The HPLC method presented here shows an easy but reliable and precise analysis of the fat-soluble vitamins retinol (A).14 0. for example. © Copyright 1998 Agilent Technologies Released 12/98 Publication Number 5968-2970E Retinol Cholecalciferol a-Tocopherol * Injection volume: 5 µl The performance of the HPLC method is shown in the table above.