Simultaneous determination of aurantio-obtusin, chrysoobtusin, obtusin and 1-desmethylobtusin in rat plasma by UHPLC-MS/MS.

A sensitive and reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous determination of four active components of Semen Cassiae extract (aurantio-obtusin, chrysoobtusin, obtusin and 1-desmethylobtusin) in rat plasma after oral administration. Chromatographic separation was achieved on an Agilent Poroshell 120 C18 column with gradient elution using a mobile phase that consisted of acetonitrile-ammonium acetate in water (30 mm) at a flow rate of 0.4 mL/min. Detection was performed by a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The calibration curve was linear over a range of 3.24-1296 ng/mL for aurantio-obtusin, 0.77-618 ng/mL for chrysoobtusin, 34.55-1818 ng/mL for obtusin and 1.86-1485 ng/mL for 1-desmethylobtusin. Inter- and intra-day assay variation was <15%. All analytes were shown to be stable during all sample storage and analysis procedures.