Whole blood concentration of chloroquine
Blood samples for the determination of chloroquine concentration were obtained from trial (iv). Approximately 250 μl arterial blood samples were drawn after 25, 45 and 60 minutes of chloroquine infusion from 6 rats for the analysis of whole blood chloroquine concentrations. Four 50 μl aliquots were accurately pipetted on to a sheet of Whatman grade 3 blotting paper, and protected from light exposure.
Standards were prepared by adding aliquots of chloroquine, giving final concentrations ranging from 0 to 30 μM, on to chloroquine-free blood spots. All samples were carefully cut from the surrounding paper, macerated and placed in individual glass vials containing 50 μl of a 1 μg ml-1 solution of 7-chloro-4-(5-diethylamino-1-methylpentyl-amino)-quinoline diphosphate to serve as an internal standard, and 3 ml of 0.2 M HCI. The vial contents were vortexed, allowed to settle, filtered and added to 0.5 ml of 5 M NaOH and 2.5 ml each of methyl-tertiary-butyl ether (MTBE) and hexane. This was centrifuged and the organic layer separated. Fresh MTBE and hexane were added to the remaining aqueous phase, and the extraction procedure repeated. The solvent was evaporated from each organic sample by heating combined with a gentle flow of dry nitrogen. Samples were then stored at -20°C.
Chloroquine was detected using an Isochrom LC Spectra-Physics pump equipped with a Rheodyne injector, a Spectra 100 fluorescence detector and a Chromjet integrator. The excitation wavelength was 340 nm and a 370 nm emission filter was used (Looareesuwan et al., 1986). The column (0.25m x 4.6 mm internal diameter) was packed with Spherisorb silica (5 μM particles; Capital HPLC) and eluted with an isocratic mobile phase consisting of acetonitrile: methanol: diethylamine (94: 5.5: 0.5), flowing at 1.5 ml min-1. The limit of detection for chloroquine was 3.1 nM and the precision of the method was 3.5% at 156 nM.