Liquid chromatographic tandem mass spectrometric assay for simultaneous quantification of 97/78 and its in-vivo metabolite 97/63, a novel trioxane antimalarial in human plasma and its application to protein binding study

Abstract

A sensitive, selective and specific LC-MS/MS assay for simultaneous quantification of 97/78 and its active in-vivo metabolite 97/63, a novel 1,2,4-trioxane antimalarial in human plasma has been developed and validated using alfa-arteether as internal standard (IS). Extraction from plasma involves simple protein precipitation method. The analytes were chromatographed on a Columbus C18 column with guard by isocratic elution with acetonitrile: ammonium acetate buffer (10mM, pH 4.0) (80:20 v/v) as mobile phase at a flow rate of 0.45 ml min-1 and analyzed by API-4000 LC-MS/MS in multiple reaction-monitoring (MRM) positive ion mode. The chromatographic run time was 4.0 min. The weighted (1/x2) calibration curves were linear over a range of 1.56-200 ng mL-1 with correlation coefficients >0.998. For both analytes, the limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.5ng mL-1 1.56ng mL-1, respectively. The recovery of 97/78, 97/63 and IS from spiked control samples were >90 persent and their matrix suppression obtained were <8 persent. The accuracy (persent Bias) and precisions (persent RSD) for both analytes were <6.78persent. Both analytes were stable after three freeze-thaw cycles (persent deviation <12.80), long-term for 30 days in plasma at −60°C (persent deviation <14.38), for 8 h on bench–top in plasma at ambient temperature (persent deviation <1.52) and also in auto-sampler for 12 h ( deviation <3.9 persent). The validated method was successfully applied to protein binding study of 97/78 and metabolite 97/63 in human plasma. Furthermore, the validated method will be applicable in various clinical phase and drug interaction studies.