Liquid chromatography–tandem mass spectrometric assay for eltrombopag in 50 μL of human plasma: A pharmacokinetic study

•First LC–MS/MS report for eltrombopag in human plasma.•Use of stable labeled isotope as IS for any variability during extraction and LC–MS/MS analysis.•The method employs only 50 μL of plasma volume using single step protein precipitation procedure.•The total analysis time is shortest.•Method reproducibility was demonstrated by incurred sample reanalysis.

Eltrombopag is a thrombopoietin receptor agonist, used in the treatment of thrombocytopenia. This paper describes a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay method for the determination of eltrombopag in human plasma samples using eltrombopag 13C4 as internal standard (IS). Analyte and the IS were extracted from 50 μL of human plasma using protein precipitation technique with no drying, evaporation and reconstitution steps. The chromatographic separation was achieved on a C18 column by using a mixture of 10 mM ammonium formate (pH 3) and acetonitrile (10:90, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The linearity of the method was established in the concentration range 50.0–10007 ng/mL with r2 ≥ 0.99. The intra-day and inter-day precision and accuracy results in four validation batches across five concentration levels were well within the acceptance limits. The proposed method was found to be applicable to pharmacokinetic studies.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide