Determination of suvorexant in human plasma using 96-well liquid–liquid extraction and HPLC with tandem mass spectrometric detection

•LC–MS/MS method for determination of suvorexant (MK-4305, Belsomra®) in human plasma.•The standard curve range was 1–1000 ng/mL.•100 μL of plasma was extracted with MTBE in the 96-well format.•Stable isotope labeled 13C2H3-suvorexant was used as an internal standard.

A method, using liquid chromatography with tandem mass spectrometric detection (LC–MS/MS), was developed for the determination of suvorexant (MK-4305, Belsomra®), a selective dual orexin receptor antagonist for the treatment insomnia, in human plasma over the concentration range of 1–1000 ng/mL. Stable isotope labeled 13C2H3-suvorexant was used as an internal standard. The sample preparation procedure utilized liquid–liquid extraction, in the 96-well format, of a 100 μL plasma sample with methyl t-butyl ether. The compounds were chromatographed under isocratic conditions on a Waters dC18 (50 × 2.1 mm, 3 μm) column with a mobile phase consisting of 30/70 (v/v %) 10 mM ammonium formate, pH3/acetonitrile at a flow rate of 0.3 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for suvorexant (m/z 451 → 186) and 13C2H3-suvorexant (m/z 455 → 190) on an Applied Biosystems API 4000 tandem mass spectrometer was used for quantitation. Intraday assay precision, assessed in six different lots of control plasma, was within 10% CV at all concentrations, while assay accuracy ranged from 95.6 to 105.0% of nominal. Quality control (QC) samples in plasma were stored at −20 °C. Initial within day analysis of QCs after one freeze-thaw cycle showed accuracy within 9.5% of nominal with precision (CV) of 6.7% or less. The plasma QC samples were demonstrated to be stable for up to 25 months at −20 °C. The method described has been used to support clinical studies during Phase I through III of clinical development.