Determination of genkwanin in rat plasma by liquid chromatography-tandem mass spectrometry: Application to a bioavailability study

Abstract

We developed and validated a sensitive, rapid, and specific liquid chromatography tandem mass spectrometry method to determine genkwanin in rat plasma. Genistein was used as the internal standard. After liquid-liquid extraction with ethyl acetate, the chromatographic separation of genkwanin was achieved by using a reversed-phase HPLC using Agela Venusil MP-C18 analytical column (2.1mm×50mm, 5μm particles) with a mobile phase of methanol (A)-water (B) (65:35, v/v) containing 5mM ammonium acetate and 0.1% formic acid. The detection was performed by negative ion electrospray ionization in multiple-reaction monitoring mode by using transitions of m/z 283.1→268.1 and m/z 269.1→133.0 for genkwanin and IS, respectively. Good linearity was observed in the concentration range of 3.84ng/ml to 3840ng/ml (r2>0.99), and the lower limit of quantification was 3.84ng/ml in 100μl of rat plasma. The intra- and inter-day accuracy and precision of genkwanin were both within acceptable limits. This present method was successfully applied to a pharmacokinetic study of genkwanin in rats following oral (50mg/kg) and intravenous (5mg/kg) administration. For the oral administration group, the maximum mean concentration of genkwanin in plasma (Cmax, 36.9±9.4ng/ml) was achieved at 3.83±1.33h (Tmax), and the area under the plasma concentration versus time curve from 0h to 12h (AUC0–12h) was 218±40ngh/ml. For the intravenous administration group, essential pharmacokinetic parameters such as Cmax (1755±197ng/ml) and AUC0–12h (2349±573ngh/ml) were shown. The result showed that the compound was poorly absorbed with an absolute bioavailability of approximately 1.1%.