Simultaneous Determination of Baicalein, Baicalin, Wogonin, and Wogonoside in Rat Plasma by LC-MS/MS for Studying the Pharmacokinetics of the Standardized Extract of Scutellariae Radix

Abstract

A new composition of standardized Scutellariae Radix extract (HPO12) was developed for treatment of Alzheimer's disease. For the preclinical pharmacokinetic study of HPO12, a rapid, sensitive, and selective LCMS/MS method was developed and validated for the simultaneous determination of 4 bioactive compounds, baicalein, baicalin, wogonin, and wogonoside. After extraction with ethylacetate, chromatographic analysis was performed on a Thermo <TEX>$C_{18}$</TEX> column (<TEX>$150mm{\times}2.1mm$</TEX>, <TEX>$3{\mu}m$</TEX>) with a mobile phase consisting of 0.1% formic acid (A) and 0.1% formic acid in 95% acetonitrile (B) by using gradient elution at a flow rate of <TEX>$250{\mu}L/min$</TEX>. Analytes introduced to a mass spectrometer were monitored by multiple reaction monitoring (MRM) in positive ion mode. Using <TEX>$25{\mu}L$</TEX> of plasma sample, the method was validated over the following concentration ranges: 25-5000 ng/mL for baicalein, 20-40000 ng/mL for baicalin, 1-1000 ng/mL for wogonin, and 5-10000 ng/mL for wogonoside. The intra- and inter-day precision and accuracy of the quality control samples at the 4 concentrations showed <TEX>$\leq$</TEX> 13.7% relative standard deviation (RSD) and 86.6-105.5% accuracy. The method was successfully applied to determine the concentrations of baicalein, baicalin, wogonin, and wogonoside in rat plasma after intraperitoneal and oral administrations of HPO12.