High-performance liquid chromatographic assay for the determination of sulfadoxine and N-acetyl sulfadoxine in plasma from patients infected with sensitive and resistant Plasmodium falciparum malaria

Abstract

A reversed-phase high-performance liquid chromatographic method using a mobile phase of acetonitrile–methanol–trifluoroacetic acid–water (16.1:7.2:0.1:76.6, v/v/v/v) at a flow rate of 1.0 ml min −1 on a LiChrospher™ RP-18 column with UV (254 nm) detection has been developed for the separation of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma. No interferences due to endogenous compounds or common antimalarial drugs were noticed. The limit of detection for sulfadoxine and N-acetyl sulfadoxine was 0.01 μg ml −1 with a signal-to-noise ratio of 5:1 while the limit of quantification was 2.5 μg ml −1. Intra-day mean relative standard deviations (RSD's) for sulfadoxine and N-acetyl sulfadoxine were 2.6 and 2.8%, respectively, while mean inter-day RSD's for sulfadoxine and N-acetyl sulfadoxine were 2.4 and 2.8%, respectively. Extraction recoveries averaged 90.6% for sulfadoxine and 86.9% for N-acetyl sulfadoxine. The method was applied for the assay of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma from Plasmodium falciparum malaria patients. Mean plasma sulfadoxine concentrations on day 2 (51 h) from samples collected from sensitive and resistant P. falciparum patients treated with three tablets of Fansidar™ were 62.8 and 60.5 μg ml −1, respectively. Mean ratio of N-acetyl sulfadoxine to sulfadoxine was 9.1% for responders and 13.9% for non-responders which revealed that higher amounts of the metabolite N-acetyl sulfadoxine were present in non-responders. The method described should find an application in the therapeutic monitoring of malaria patients.