Novel LC–MS/MS method for analyzing imperialine in rat plasma: Development, validation, and application to pharmacokinetics

• We developed and validated a novel LC–MS/MS method for imperialine quantification.
• The method debuted on pharmacokinetic study of imperialine in rats.
• Pharmacokinetic study was done at low doses (mg/kg, i.v., 5; p.o., 1, 5 and 10).
• Oral bioavailability of 3 p.o. groups was 31.2%, 53.6% and 47.4%, respectively.

A sensitive, selective, rapid liquid chromatography–electrospray ionization-tandem mass spectrometric method was developed and validated in rat plasma to quantify imperialine, a major active constituent extracted from Bulbus Fritillariae Cirrhosae. Before analysis, plasma samples were pre-treated using cost-effective protein precipitation in order to extract imperialine and the internal standard, carbamzepine. The two analytes were then separated on a Diamonsil ODS chromatography column using gradient elution with a mobile phase of 0.1% aqueous formic acid and acetonitrile. Mass spectrometry was carried out in multiple reaction monitoring mode using a positive electrospray ionization interface. The calibration curve was linear (r2 = 0.9998) over the concentration range 2–1000 ng/mL, while the validated limit of determination (LOD) was 0.5 ng/mL. Precision varied from 0.1% to 7.1%, and the accuracy varied within ±2%. The method proved robust to sample freezing and thawing, as well as short- and long-term sample storage. The developed method was successfully applied to the pharmacokinetic study of imperialine in rats. Different amounts of imperialine were administered in single doses orally or through the caudal vena cava, and pharmacokinetic parameters were evaluated. Oral bioavailability with a dose of 1 mg/kg was 31.2%; 5 mg/kg, 53.6%; and 10 mg/kg, 47.4%.