Quantification of Lumefantrine in Human Plasma Using LC-MS/MS and Its Application to a Bioequivalence Study

An analytical method based on protein precipitation has been developed and validated for analysis of lumefantrine in human plasma. Artesunate was used as an internal standard for lumefantrine. Inertsil ODS column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode using an API-3000 system. The total run time was 2.5 minutes. The proposed method has been validated with linear range of 200–20000？ng/mL for lumefantrine. The intrarun and interrun precision values are within 6.66% and 5.56%, respectively, for lumefantrine at the lower limit of quantification level. The overall recovery for lumefantrine and artesunate was 93.16% and 91.05%, respectively. This validated method was used successfully for analysis of plasma samples from a bioequivalence study. 1. Introduction Lumefantrine (IUPAC name: 2-(dibutylamino)-1-[(9Z)-2,7-dichloro-9-(4-chlorobenzylidane)-9H-fluoren-4-yl]ethan-1-ol) is an antimalarial agent used to treat acute uncomplicated malaria. It is administered in combination with artemether for improved efficacy. This combination therapy exerts its effects against the erythrocytic stages of Plasmodium and may be used to treat infections caused by P. falciparum and unidentified plasmodium species, including infections acquired in chloroquine-resistant areas [1]. Few bioanalytical methods are reported to determine lumefantrine in plasma using HPLC-UV [2–4] and LC-MS/MS [5–8] detection. All these reported methods require total run time ranging from 5 to 17？min. Pharmacokinetic study in five healthy volunteers under fasting condition was studied by Cesar et al. [8]. Bioequivalence study with comparative safety evaluation was conducted on 72 healthy Indian human subjects under a fed condition by Khandave et al. [6]. All reported methods have long run time. Hence, it felt necessary to develop and validate a rapid and selective method that can be successfully applied to a bioequivalence study. In the present paper we would like to present a simple and high-throughput protein precipitation method for quantification of lumefantrine using artesunate as an internal standard with LC-MS/MS detection. The application of this validated method in analyzing samples from a bioequivalence study involving lumefantrine is also presented. 2. Experimental 2.1. Chemicals and Reagents The reference standard of lumefantrine was provided by Ipca Laboratories Ltd. (Mumbai, India). The reference standard