Sensitive high performance liquid chromatographic method for the determination of proguanil and its metabolites, cycloguanil and 4-chlorophenylbiguanide in biological fluids

Abstract

A new simple, sensitive, cost-effective and reproducible high performance liquid chromatographic (HPLC) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 4chlorophenylbiguanide (4-CPB) in urine and plasma is described. The extraction procedure is a simple three-step process that has eliminated the need for costly extraction and evaporation equipment. The mobile phase consisted largely of buffer, making the method cheap to run. The calibration plots were linear over the concentration range up to 3.0 µg/ml PG, CG and 4-CPB in urine and concentration range up to 1000 ng/ml in plasma. The correlation coefficients (r) were of the order of 0.99 and above for PG and 4-CPB and 0.98 for CG. The ion pair method was carried out on a 5 µ reversed-phase C-18 column, using perchlorate ion as the counter ion and ultra violet detection at 254 nm. The method was reproducible with coefficient of variation for PG, CG and 4-CPB, being less than 10% in urine and plasma. PG was well resolved from its metabolites, CG and 4-CPB, and the internal standard, pyrimethamine. The limit of detection of PG was 10 ng/ml and the recovery was greater than 90% in urine and plasma. The analytical method therefore, exhibits good precision and sensitivity and is one of the few methods that can detect PG and the two metabolites CG and 4-CPB. The analytical method developed in this study was used to determine PG bioavailability after rectal administration in humans.