Sample Extraction (Liquid Liquid Extraction Method)
Before extraction, bulk spiked
Calibration curve standards (
CC) and Quality control (QC) samples
were removed from the deep freezer and thawed at ro
om temperature. Calibration standards and
Quality control samples were then made ready for ex
traction in 4 mL polypropylene tubes.
Exactly 50
μ
L of plasma was pipette out into prelabelled polypr
opylene tubes, to this 25
μ
L of
internal standard dilution (2000 ng/mL) was added a
nd vortex for 20 seconds. 2 mL of extraction
solvent was added [Diethyl ether: Isopropyl alcohol
] (1:1 v/v) and vortexed for 3 minute.
Samples were centrifuged at 2000 RPM at 4°C for 2 m
inutes and the 1.9 mL supernatant layer
was separated, evaporated to dryness under nitrogen
gas stream, reconstituted 1 mL of dilution
solvent and these samples were transferred to the a
uto sampler vial and 5
μ
L was injected into
the chromatographic system