Liquid chromatographic tandem mass spectrometric assay for quantification of 97/78 and its metabolite 97/63: A promising trioxane antimalarial in monkey plasma

The present manuscript describes development and validation of LC-MS/MS assay for the simultaneous quantitation of 97/78 and its active in-vivo metabolite 97/63 in monkey plasma using α-arteether as internal standard (IS). The method involves a single step protein precipitation using acetonitrile as extraction method. The analytes were separated on a Columbus C18 (50 mm × 2 mm i.d., 5 μm particle size) column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 4, 10 mM) (80:20 v/v) at a flow rate of 0.45 mL/min, and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) positive ion mode. The chromatographic run time was 4.0 min and the weighted (1/x2) calibration curves were linear over a range of 1.56-200 ng/mL. The method was linear for both the analytes with correlation coefficients >0.995. The intra-day and inter-day accuracy (% bias) and precisions (% RSD) of the assay were less than 6.27%. Both analytes were stable after three freeze-thaw cycles (% deviation <8.2) and also for 30 days in plasma (% deviation <6.7). The absolute recoveries of 97/78, 97/63 and internal standard (IS), from spiked plasma samples were >90%. The validated assay method, described here, was successfully applied to the pharmacokinetic study of 97/78 and its active in-vivo metabolite 97/63 in Rhesus monkeys.