Determination of suvorexant in human plasma using 96-well liquid-liquid extraction and HPLC with tandem mass spectrometric detection.
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 1-1000ng/mL. Stable isotope labeled (13)C(2)H3-suvorexant was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction, in the 96-well format, of a 100μL plasma sample with methyl t-butyl ether. The compounds were chromatographed under isocratic conditions on a Waters dC18 (50×2.1mm, 3μm) column with a mobile phase consisting of 30/70 (v/v %) 10mM ammonium formate, pH3/acetonitrile at a flow rate of 0.3mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for suvorexant (m/z 451→186) and (13)C(2)H3-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.
Pharmacokinetics Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, WP75B-300, West Point, PA 19486, USA. Electronic address: sheila_breidinger@merck.com.
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Breidinger
Pharmacokinetics Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, WP75B-300, West Point, PA 19486, USA.
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Simpson
Pharmacokinetics Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, WP75B-300, West Point, PA 19486, USA.
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Mangin
Pharmacokinetics Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, WP75B-300, West Point, PA 19486, USA.
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Woolf
D001381:Azepines / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D059625:Liquid-Liquid Extraction / Q000379:methods*; D000068776:Sleep Aids, Pharmaceutical / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014230:Triazoles / Q000097:blood*
D001381:Azepines; D000068776:Sleep Aids, Pharmaceutical; D014230:Triazoles; C551624:suvorexant
Bioanalytical; Human plasma; LC–MS/MS; Liquid–liquid extraction; Suvorexant