A volume of 100 mL methanol was transferred into the
Erlenmeyer flask containing (50 g) fortified soil sample
and allowed to stand for 2 h. The Erlenmeyer flask was
placed onto orbital shaker for 30 min. After shaking, the
solutions were filtered into the round bottom flask of
500 mL capacity through Whatman filter paper No.1
bearing a bed of anhydrous sodium sulphate. Solvent was
removed using vacuum evaporator. The residual cake was
re-extracted twice with additional volume of 50 mL
methanol. The methanol extracts were collected, pooled
and concentrated to smaller volume (5–10 mL) using
vacuum evaporator at B40C. The concentrated extract
was subjected to further clean up by column chromatography.
A glass column packed with florisil as adsorbent
placed in between two layer of anhydrous sodium sulphate
was employed. The column was pre-conditioned with
methanol and concentrated extracts were loaded onto top of
the column and eluted with 100 mL acetonitrile @ 2 mL/
min. Eluate was concentrated to dryness using rotary
vacuum evaporator at B40C and residue re-dissolved in
5 mL acetonitrile. The samples were transferred into volumetric
flask 10 mL capacity using Whatman No. 1 filter
paper and final volume was made up to the mark with
acetonitrile.