HPLC was performed with a Waters (USA) system incorporating
an Alliance 2690 separations module and a 996 photodiode-array detector
and operated by Millennium32 software. Tocopherols were separated on a
Waters Symmetry C18 column (3.9 mm × 150 mm) with a Waters Symmetry
C18 (3.9 mm × 10 mm) guard column. The mobile phase was initially a
linear gradient of acetonitrile in water (from 95 to 100% in 10 min) then
100% of acetonitrile for 10 min; the flow rate was 1 mL min−1
. The column
temperature was 45°C and the wavelength-range scanned was 275–
350 nm. Samples were kept in an autosampler at 30°C and the injection
volume was 25 µL. Peak areas of tocopherols were integrated at 295 nm.
Tocopherols were identified by coelution after spiking the extracted samples
with solutions of the tocopherols in ethanol. To ensure reproducible
retention on the column, after each batch of 30–40 milk extracts the column
was washed with propan-2-ol (1 mL min−1
for 60 min at 45°C).