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The mixture was gently dried in a rotary evaporator followed by high vacuum treatment to remove all solvent.
3 N aqueous HCl solution (10 ml) was added, and the mixture was gently heated to 50 °C for 30 min.
Thirty minutes after mixing, the mixture was gently centrifuged to obtain the two-phase separations.
The mixture was gently stirred at 4 °C for 1 h.
The reaction mixture was gently refluxed and stirred for 24 h.
The mixture was gently agitated overnight at 4°C on a shaker.
After cooling, 2.5 mL of 70%% HClO4 was added, and the mixture was gently boiled until dense white fumes appeared.
The above reaction mixture was gently mixed and incubated for 3 h with gentle and continuous vortexing.
Then equal volume of chloroform: isoamyl alcohol (24:1 v/v) was added and mixture was gently mixed for 10 min by inverting the tube.
The mixture was gently heated on a hot plate after a few boiling chips had been added and evaporated to about 20 mL.
The mixture was gently boiled in a hot plate until about 5 10 ml of extract remains in the flask and allowed to cool for about 15 min.
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