New liquid chromatographic method for simultaneous quantification of Atovaquone and Proguanil with its active metabolite Cycloguanil in human plasma

Objective: The aim of the present study is to develop HPLC method for simultaneousquantification of atovaquone and proguanil with its active metabolite cycloguanilin human plasma. Methodology: A specific and accurate highperformance liquid chromatographic method has been developed using Phenyl (150×4.6 mm, 5 μm) column maintained at 18 °C. The separation was achieved using a mobile phase composed of phosphate buffer pH 7.2 and methanol (45:55%). The mobile phase was maintained at flow rate of 0.8 mL/min. The analytes were monitored at 254 nm usingultra-violet detector. The plasma samples extractions was carried out using tert-Butyl Methyl Ether: Dichloromethane (80:20% v/v) mixture. Tramadol was used as an internal standard (ISTD). Result: The developed method was validated as per US FDA guidelines and found to be highly specific, precise and accurate. Moreover, atovaquone, proguanil and cycloguanilwere stable in plasma at various stability conditions. Conclusion: The developed method is simple, economic, and can be used for quantification of said drugs human plasma samples.