Validation of an UHPLC–MS/MS Method for the Determination of Glaucocalyxin A, a Novel Potent Negative Akt Regulator in Rat Plasma, Lung and Brain Tissues: Application to a Pharmacokinetic Study

Glaucocalyxin A, a novel potent negative Akt regulator, is a major active constituent of Rabdosia japonica. A simple, specific and sensitive ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the quantification of glaucocalyxin A in rat plasma, lung and brain tissues after intravenous administration. Sample preparation was carried out through a simple liqiud-liquid extraction with ethyl acetate using bullatine A as internal standard. The chromatographic separation was achieved by using an Agilent ZORBAX Eclipse Plus C18 column (3.0 mm x 100 mm, 1.8 mum) with a mobile phase of acetonitrile and water containing 0.1% formic acid in a gradient elution. Mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring transitions at m/z 333.2 --> 157.1 for glaucocalyxin A and m/z 344.2 --> 128.1 for IS. Calibration curves were linear over the ranges of 20.0-4000 ng/mL for both plasma and tissue samples (r2 > 0.99). The Lower limit of quantification (LLOQ) was 0.284 ng/mL. The intra-day and inter-day precision relative standard deviation (RSD%) were <14.9%, while the accuracy ranged from -12.5 to 17.0% for LLOQ and quality control samples. This UHPLC-MS/MS method was successfully applied in the pharmacokinetics, lung and brain tissue distributions of glaucocalyxin A after intravenous administration.