A representative 20 g portion of previously homogenized
samples (either bitter gourd or soil) was weighed into a 100 mL
centrifuge tube. The samples were fortified with a suitable volume
of standard solution, mixed well, and equilibrated for 1 h. Then
40 mL of chromatographically pure ethyl acetate was added, and
the tube was capped. The mixture was vortexed for 1 min. The procedure
for soil was identical throughout, except for the previous
step, where 10 mL of distilled water was also added to the soil.
Then, the tube was centrifuged for 10 min at 4000 rpm. About
20 mL of the upper ethyl acetate layer was concentrated to dryness
on a rotary evaporator under a vacuum and added 2 mL of
n-hexane/ethyl acetate (9:1, v/v), then cleaned up with glass column
of florisil and a solid-phase extraction (SPE) column of NH2,
respectively.