Validation of a method for the simultaneous quantification of chloroquine, desethylchloroquine and primaquine in plasma by HPLC-DAD

•Chloroquine, desethyl chloroquine and primaquine level were quantified simultaneously by HPLC-DAD.•The method validation parameters were adequate according international standards.•Sample preparation procedure is easy and inexpensive.•The method was successfully applied to plasma patient samples.•The method is suitable to carry out therapeutic efficacy studies. One of the most important aspects regarding the therapeutic efficacy of antimalarials is its quantification in biologic fluids. The detection and measurement of antimalarial drug levels is important for demonstrating (1) adequate absorption of the drug being given, (2) compliance in taking the full regimen required for treatment and (3) the level of drug in the blood at any time during the test period that parasites reappear. There is a lack of validated methods that simultaneously quantify different antimalarials administered at the same time, such as the use of chloroquine (CQ) and primaquine (PQ) in infections caused by Plasmodium vivax. In this study, a bioanalytical method was validated for the simultaneous quantification of primaquine (PQ), chloroquine (CQ) and desethylchloroquine (DSCQ) in human plasma using liquid–liquid extraction and high performance liquid chromatography with a diode array detector (HPLC-DAD). The PQ was evaluated over a concentration range of 100–3000nM and the CQ and DSCQ was evaluated over a concentration range of 20–2000nM. The selectivity of the method was verified by checking for interference by commonly used antimalarials and plasma samples. The accuracy and precision of the method was assessed for drugs spiked into human plasma and recoveries of 83.7%, 92.3%, and 76.5% were obtained for CQ, DSCQ, and PQ, respectively. The applicability of this method was also demonstrated with blood samples from patients with vivax malaria that received combination CQ plus PQ treatment. The simultaneous detection and accurate measurement of CQ, DSCQ, and PQ levels in human plasma provides an important and economical method for validating and monitoring sensitivity/resistance of P. vivax to more common treatment regimen.