Metabolite Analysis

Blood was collected in a heparin tube and centrifuged for 5 min at 5000 rpm. Plasma was separated from blood cells, and about 1 ml was diluted with 2 ml of 0.1 M hydrochoric acid and loaded onto a tC18 Sep-Pak cartridge, which was pre-activated by elution with 6 ml of methanol and 12 ml of water, respectively. The cartridge was washed with 3 ml water to collect the polar radioactive fraction. Thereafter, the tC18 Sep-Pak cartridge was eluted with 1 ml of methanol and 2 ml of water to collect the mixture of non-polar metabolites. This fraction was further analysed by HPLC on a Dionex Ultimate 3000 system (Dionex, Sunnyvale, CA, USA) and equipped with a 1-ml loop. As a stationary phase, a Phenonenex Gemini C18, 250 × 10 mm, 5 μm (Phenomenex, Torrance, CA, USA) was used. The mobile phase consisted of 75 % 0.1 % trifluoroacetic acid in water in acetonitrile. The eluent was collected with a fraction collector (Teledyne ISCO Foxy Jr., Lincoln, NE, USA), and the fractions were counted for radioactivity with a Wallac 1470 gamma counter (Perkin Elmer, Waltham, MA, USA).