Simultaneous determination of macitentan and its active metabolite in human plasma by liquid chromatography-tandem mass spectrometry

• A method was developed for simultaneous determination of macitentan and its active metabolite.
• The developed method was fast, sensitive, and reliable.
• The method was successfully applied to a pharmacokinetic study.

Macitentan is a newly approved endothelin receptor antagonist (ERA) for the long-term treatment of PAH with superior receptor-binding properties and a longer duration of action compared to other available ERAs. However, analytical methods for simultaneous determination of macitentan and its active metabolite, ACT-132577, in human plasma have not been fully reported in the literature. In this work, a fast, sensitive, and reliable high-performance liquid chromatography-tandem mass spectrometry method (HPLC–MS/MS) was firstly developed and completely validated for simultaneous determination of macitentan and its active metabolite in human plasma. Plasma samples were processed with a protein precipitation using acetonitrile, followed by chromatographic separation using an Inertsil ODS-SP column (100 × 2.1 mm, 3.5 μm) under isocratic elution with a mobile phase consisting of acetonitrile and 0.2% formic acid at a flow rate of 0.3 mL/min. Quantification was operated in multiple reaction monitoring (MRM) mode using the transitions m/z 547.1 → 201.0 for macitentan, m/z 589.0 → 203.0 for ACT-132577, and m/z 380.5 → 243.3 for the IS (donepezil). The assay exhibited a linear range of 1–500 ng/mL for both macitentan and ACT-132577. The accuracy and the intra- and inter-precisions were within acceptable ranges and no significant matrix effect was observed during the method validation. The developed method was successfully utilized to a human pharmacokinetic study of macitentan as well as ACT-132577 after oral administration of 10 mg macitentan tablet in healthy Chinese volunteers.