Determination of N-methylcytisine in rat plasma by UPLC-MS/MS and its application to pharmacokinetic study

•First report of development an UPLC-MS/MS method for the determination of N-methylcytisine in rat plasma.•First report of pharmacokinetic study of N-methylcytisine after oral and intravenous administration.•First report of the bioavailability of N-methylcytisine.

In this work, a sensitive and selective UPLC-MS/MS method for determination of N-methylcytisine in rat plasma is developed. After addition of hordenine as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 μm) with acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 205.1→58.0 for N-methylcytisine, and m/z 166.1→121.0 for IS. Calibration plots were linear throughout the range 2-2000 ng/mL for N-methylcytisine in rat plasma. Mean recoveries of N-methylcytisine in rat plasma ranged from 86.1% to 94.8%. RSD of intra-day and inter-day precision were both < 13%. The accuracy of the method was between 94.5% and 109.4%. The method was successfully applied to pharmacokinetic study of N-methylcytisine after either oral or intravenous administration. For the first time, the absolute bioavailability of N-methylcytisine was reported as high as 55.5%.