Establishment of a simultaneous assay for lemborexant, a novel dual orexin receptor antagonist, and its three metabolites, and its application to a clinical protein binding study.

A simultaneous assay for the determination of lemborexant and three metabolites (M4, M9, and M10) in human plasma and phosphate buffered saline (PBS) was developed and validated using liquid chromatography with tandem mass spectrometry, in support of plasma protein binding (PPB) studies. The analytes were extracted from plasma and PBS by solid phase extraction and then chromatographed on a reversed phase C18 column to ensure peak separation of three metabolites with the same mass transition. The analytes and the corresponding deuterated substances used as an IS were detected in the positive ion mode by multiple reaction monitoring. Lemborexant and three metabolites were quantifiable from 4 and 300 pg/mL in PBS and plasma, respectively, without any carryover. Extraction recovery was almost complete without matrix effects. The accuracy and precision in the intra- and inter-assay reproducibility were within the criteria as well as in the dilution integrity. Stability of the four analytes was ensured to cover duration of equilibrium dialysis and storage of samples. The established method was applied to an ex vivo PPB study in humans.