In this experiment, we will be using  liquid–liquid extraction with hexane after simple saponification for the extraction of vitamin E from human milk.

 For this analysis, High Performance Liquid Chromatography (HPLC) with UV detection will be used.


Chromatographic System and Conditions:


1. Waters Symmetry C18 guard column (3.9mm x 10mm)
2. Waters Symmetry C18 analytical column (3.9mm x 150mm)
3. Gradual change of mobile phase
      - First 10 minutes: Linear gradient of acetonitrile in water (from 95% - 100%)
      - Subsequent 10 minutes: 100% acetonitrile 
4. Column temperature: 45 Degree Celsius
5. Flow rate: 1mL/min
6. Wavelength range used to scan: 275nm - 350nm 
7. Injector volume: 25 µL

* Samples were placed & stored in autosampler @ 30 degree Celsius

After 30-40 milk extracts analysis, analytical column is washed with propan-2-ol under the following conditions:
- Flow rate of 1mL/min for 60 minutes 
- Temperature of 45 degree Celsius. 

*This is to ensure reproducible retention on the column.

Evaluation the recovery of the δ-tocopherol spike after extraction was done using:
- 10 μL spiking solution that was diluted with 0.5 mL methanol–propan-2-ol (1:1, v/v) 
- 25 μL of this solution was injected on the column.   

- Peak areas of δ-tocopherol spikes in milk extracts were expressed as percentages of the peak areas of δ-tocopherol solutions prepared. 
- The amounts of native α and γ-tocopherols were calculated on the basis of their peak areas and the percentage recovery. 
- The latter was calculated for each sam-ple using the peak area of δ-tocopherol spike. 

Regression analysis was performed using Minitab software. The difference was considered significant if P < 0.05. Means and standard deviations are reported as well.