Simultaneous analysis of avatrombopag, eltrombopag, and hetrombopag in human plasma by UPLC-MS/MS for therapeutic drug monitoring

Avatrombopag, eltrombopag, and hetrombopag are three oral thrombopoietin receptor agonists used to treat thrombocytopenia. To investigate the pharmacokinetic properties, a fast and robust UPLC-MS/MS method was developed and validated for simultaneous determination of avatrombopag, eltrombopag, and hetrombopag in human plasma. Chromatographically separation was achieved on an Acquity BEH C18 column using a gradient elution of methanol-water (20:80, v/v) and acetonitrile containing 0.1% formic acid at a flow rate of 0.4 mL/min within 4.5 min. The linearity was assessed over the range of 7.5-1500 ng/mL for avatrombopag, 50-10,000 ng/ mL for eltrombopag, and 5-1000 ng/mL for hetrombopag. This method was validated with adequate results in terms of selectivity, precision (intra-and inter-day CV from 4.2% to 12.2%), accuracy (RE from-6.6% to 7.5%), dilution integrity, carryover, recovery, matrix effects, as well as stability of the analytes under various conditions. The developed approach has been successfully applied to the therapeutic drug monitoring of avatrombopag, eltrombopag, and hetrombopag in patients with immune thrombocytopenia.