LC-MS/MS method for simultaneous determination of ramelteon and its metabolite M-II in human plasma: Application to a clinical pharmacokinetic study in healthy Chinese volunteers.

A sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of ramelteon and its active metabolite M-II in human plasma. After extraction from 200 µL of plasma by protein precipitation, the analytes and internal standard (IS) diazepam were separated on a Hedera ODS-2 (5 µm, 150 × 2.1 mm) column with a mobile phase consisted of methanol-0.1% formic acid in 10 mm ammonium acetate solution (85:15, v/v) delivered at a flow rate of 0.5 mL/min. Mass spectrometric detection was operated in positive multiple reaction monitoring mode. The calibration curves were linear over the concentration range of 0.0500-30.0 ng/mL for ramelteon and 1.00-250 ng/mL for M-II, respectively. This method was successfully applied to a clinical pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ramelteon. The maximum plasma concentration (Cmax ), the time to the Cmax and the elimination half-life for ramelteon were 4.50 ± 4.64ng/mL, 0.8 ± 0.4h and 1.0 ± 0.9 h, respectively, and for M-II were 136 ± 36 ng/mL, 1.1 ± 0.5 h, 2.1 ± 0.4 h, respectively.