Aim: To develop a sensitive and rapid LC/MS/MS method for the determination of wogonin, an active flavonoid shown to have an inhibitory effect on the proliferation of a carcinoma cell line, in rat plasma after an oral administration.
Methods: Wogonin and daidzein (internal standard) were extracted from plasma directly with n-hexane-diethyl ether (1:4). After liquid-liquid extraction, the analytes of interest were separated on a Diamonsil C18 column. The mobile phase was acetonitrile-water-formic acid (80:20:1) with a flow rate of 0.8 mL.min-1. A Finnigan TSQ (triple stage quadruple) tandem mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source was used as detector and was operated in positive ion mode. Selected reaction monitoring (SRM) was used and transitions selected for quantitation were: m/z 284.8-->269.5 for wogonin and m/z 254.7-->198.5 for daidzein. The mass spectrometric conditions were as follows: The temperatures of the vaporizer and heated capillary were 450 degrees C and 250 degrees C, respectively. The corona discharge current was 4.00 microA. Nitrogen was used as the sheath and auxiliary gas, whose settings were 0.6 MPa and 3 mL.min-1, respectively. Argon was used as the collision gas at a pressure of 1.4 Pa. The collision energy of 35 V was chosen for both wogonin and daidzein.
Results: The calibration curve was linear over the concentration range of 0.25 to 20.0 ng.mL-1. The limit of quantitation was 0.25 ng.mL-1. Within-day and between-day precision expressed by relative standard deviation (RSD) was 2.2% to 13.1% and 5.9% to 7.3%, respectively, and the accuracy expressed by RE was -0.3% to 1.3%.
Conclusion: This method proved to be specific, accurate and sensitive enough to be applied to the pharmacokinetic studies of wogonin in rats after a single dose of 5 mg.kg-1 by oral administration.