Validation of an HPLC–MS-MS Assay for Determination of Morellic Acid in Rat Plasma: Application to Pharmacokinetic Studies

A selective and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) was developed for the quantification of morellic acid in rat plasma. HPLC was performed using a Capcell MG C18 (50 × 4.6 mm, i.d., 5 µm) column, and isocratic elution with water-acetonitrile (20:80, v/v) at a flow rate of 0.5 mL/min. Sample preparation of analyte and internal standard (gambogic acid) involved liquid-liquid extraction using ethyl acetate-isopropanol (1:1, v/v) from 50 µL plasma. The precursor → production transitions for analyte and IS were m/z 559.4 → 471.3, and m/z 627.3 → 583.3, respectively, and were monitored on a triple-quadrupole mass spectrometer, operating in negative ion scan mode. The method was validated across the dynamic concentration range of 20-7,500 ng/mL for morellic acid, with a fast run time of 6.0 min. The analytical method measured concentrations of morellic acid with accuracy (% bias) of ≤6.4% and precision (% RSD) of ≤14.0%. Morellic acid was stable during the battery of stability studies. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic studies in Sprague-Dawley rats. This method will therefore be useful for further preclinical and clinical pharmacokinetic studies of morellic acid.