Development and validation of a sensitive LC-MS-MS assay for the quantification of betulonic acid in rat plasma

A rapid, sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of betulonic acid in rat plasma. The plasma samples were prepared by protein precipitation. Chromatographic separation was achieved on an Agela MG-C18 analytical column (50 × 2.1 mm, 5 µm) using methanol-water-formic acid (80:20:0.1, v/v/v) as the mobile phase with a constant flow rate of 0.6 mL/min. Mass detection was conducted by electrospray ionization in positive ion multiple reaction monitoring mode. The calibration curves were linear over a concentration range of 3.00-3,000 ng/mL for the analyte. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. The method was successfully applied for the estimation of betulonic acid in rat plasma from a preclinical pharmacokinetics study.