Development and validation of a UHPLC–MS/MS method for the simultaneous determination of five bioactive flavonoids in rat plasma and comparative pharmacokinetic study after oral administration of Xiaochaihu Tang and three compatibilities

Abstract

An accurate, rapid, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of baicalin, wogonoside, baicalein, wogonin, and oroxylin A in rat plasma. Then, the stability of baicalin and baicalein in the preparation of plasma sample was systematically investigated. The Waters BEH C column was used with a gradient mobile phase system of acetonitrile and water containing 0.1% formic acid. The analytes were detected in the multiple reaction monitoring mode with positive electrospray ionization. 100 μL fresh plasma was added with 50 μL antioxidant reagent (1 mol/L HCl containing 0.5% Vitamin C), and liquid-liquid extraction with ethyl acetate was used to extract the analytes from plasma. Lower limits of quantification of baicalin, wogonoside, baicalein, wogonin, and oroxylin A were 21.9, 4.80, 1.20, 0.848, and 0.800 ng/mL, respectively. The mean extract recoveries of five flavonoids were 69.1∼89.2%, and the precision and accuracy were within the acceptable limits. This method was further successfully applied to the comparative pharmacokinetic study of these five flavonoids in rats after oral administration of Xiaochaihutang and three compatibilities. The obtained results may be helpful to reveal the mechanism of Xiaochaihutang formula compatibility.

Pharmacokinetics of B-Ring Unsubstituted Flavones

“…In the case of GL-V9, although a concentration peak was seen after 0.5 h of oral administration, a second twin peak was recorded after 12 h (in rats), most likely related to the enterohepatic, enteric, or local recycling [60]. A double peak was detected in the plasma concentration–time curves for baicalin, oroxin A (baicalein-7- O -glucoside), baicalein, wogonoside, wogonin, and chrysin when administered in rats, related to enterohepatic, local, or enteric recycling [16,23,32,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100]. In one study in rats, it was claimed that this bi-modal “behavior” was not confirmed experimentally [101], but the authors only measured plasma samples up to 180 min, whereas in order to observe the second peak (at its maximum) at least 6–8 h would often be needed [102,103,104,105], although in other cases a second peak was reported after only two to three hours [106,107,108,109]; in dogs, following i.v.…”

“…Oxymatrine, by contrast, had no influence on the pharmacokinetics of baicalin [255]. The pharmacokinetic parameters of baicalein, wogonin, and oroxylin A also vary across other different herbal preparations [92,94,256,257,258,259], depending on their complex matrices of ingredients. It is likely that such reciprocal influences contribute to further variability of the PK of these flavones.…”