ere transferred
to 1.5 ml Eppendorf tubes. Thirty microlitres of internal standard
(IS) working solution in water (100 g/ml) were added
to each tube. One drop of 10 M phosphate buffer (pH 2.7) was
added and the tubes were vortex mixed for 3 min. Five hundred microlitres
of ice cold acetonitrile were added using a glass syringe. The tubes were vortex mixed for a further 5 min. The samples
were centrifuged at 3500 × g for 5 min. The supernatant was poured
into 8 ml silanised glass tubes. The silanised tubes were placed in
a Turbovap Concentrator
®
with the water bath set at 50
C for 20 min. The dried residue was reconstituted with 100 l mobile phase, vortex mixed for 3 min and re-centrifuged at 15,000 rpm for 3 min. Fifty microlitres of the clear supernatant were injected into the HPLC unit