High-performance liquid chromatographic determination of primaquine and carboxyprimaquine concentrations in plasma and blood cells in Plasmodium vivax malaria cases following chronic dosage with primaquine

A reversed-phase HPLC method using acetonitrile-methanol-1 M perchloric acid-water (30:9:1:95, v/v) at a how-rate of 1.5 ml/min on a mu-Bondapak C-18 column with UV detection at 254 nm was developed for the separation of primaquine, its major metabolite carboxyprimaquine and other metabolites such as N-acetyl-primaquine, 4-hydroxyprimaquine, 5-hydroxyprimaquine, 5-hydroxy-6-methoxyprimaquine, demethylprimaquine and 6-methoxyprimaquine, and also other antimalarials. The calibration graphs were linear in the range 0.025-100 mu g/ml for primaquine and 4-1000 mu g/ml for carboxyprimaquine. The within-day and day-to-day coefficients of variation averaged 3.65 and 6.95%, respectively, for primaquine and 3.0 and 7.52%, respectively, for carboxyprimaquine in plasma. The extraction recoveries for primaquine and carboxyprimaquine were 89 and 83%, respectively. The mean carboxyprimaquine concentration was much higher in plasma and blood cells of Plasmodium vivax patients than that in plasma from healthy subjects. The carboxyprimaquine level was also higher in blood cells than plasma whereas the primaquine concentration was the same in both cases.