2.4.2. Vitamin A and E
Vitamin A and E were determined simultaneously through
HPLC by following the method of Paixao and Campos
(2003). Thirty grams of milk sample was mixed with 7 g of
KOH in a 100 mL amber glass flask. Sponification of the sample was done by adding one gram each of sodium sulfide and
sodium (L) ascorbate. After sponification, sodium dodecyl sulfate was added and mixed well. Extraction of aliquot was carried out in an extrulent cartridge. The extracted sample was
evaporated under nitrogen stream and the content was diluted
with mobile phase (one percent solution of 2-propanole in nhexane). Prepared sample was analyzed for vitamin A and E
by HPLC (PerkinElmer, Series 200, USA) containing C18 column (250 · 4.6 mm, 5.0 lm particle size) and an autosampler.
Sample amount 20 lL and column temperature 25 C were
kept during HPLC analysis. During vitamins’ quantification,
mobile phase one percent solution of 2-propanole in n-hexane
was used with a flow rate of 1 mL/min. Quantification of vitamin E and A were carried out by UV detector at 292 and
326 nm, respectively.