Quantitative determination of ginsenoside Rh₂ in rat biosamples by liquid chromatography electrospray ionization mass spectrometry

Ginsenoside Rh₂ is a "hot" natural compound with great potential as a new anti-cancer drug based on abundant pharmacological experiments. However, no systemic pharmacokinetic study of Rh₂ was reported because current analysis methods could not fully meet the requirements. Thus, we developed a simple LC/MS method with highly improved sensitivities for the determination of Rh₂ in rat plasma, bile, urine, feces and most tissues. The tissues and feces were firstly homogenized mechanically using buffer and methanol as the media, respectively. Plasma, bile, urine and tissue homogenates were extracted with diethyl ether for sample preparation. Feces homogenates were directly deproteinized with acetonitrile. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer), with an ODS column (150 mm∈×∈2.0-mm i.d., 5 μm) plus a C18 guard column for separation and ammonium chloride (500 μmol) as mobile phase additive. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M∈+∈Cl]^- of Rh ₂ at m/z 657.35 and internal standard digitoxin at m/z 799.55 were monitored in selective ion monitoring mode of negative ions. The method was validated to be accurate, precise and rugged with good linearity in all matrices, according to the FDA guidelines. The lower limits of quantitation in rat plasma, urine and feces were 0.2, 0.2 and 20 ng/mL respectively. Stability studies were also performed, indicating that there were no stability-related problems in the analytical procedure of Rh₂. The proposed method was successfully applied to the preclinical pharmacokinetic research of Rh ₂ in rats, including plasma kinetics, tissue distribution and excretion studies.