LC-MS/MS for determination of aesculetin in rat plasma and its application to a pharmacokinetic study

Aesculetin, a coumarin compound present in the sancho tree and chicory, exhibits excellent antioxidant and anti-inflammatory activities in the vascular and immune system. In this study, a rapid and sensitive ultra-high performance liquid chromatography electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method was established and validated for the determination of aesculetin in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was performed on an Acquity UPLC HSS T3 C₁₈ column (2.1 × 100 mm, 1.8 μm) with gradient elution at a flow rate of 0.3 ml/min, using mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Aesculetin and puerarin (internal standard) were detected by multiple reaction monitoring in negative ion mode. The method was fully validated according to the US Food and Drug Administration guidelines. The calibration curve was linear over the range of 2-1,000 ng/ml with correlation coefficient >0.9980. The carry-over, matrix effect, extraction recovery, dilution effect, intra- and inter-day precision and the accuracy were within acceptable limits. The method was then applied to a pharmacokinetic study of aesculetin in rats. After oral administration at doses of 5, 10 and 20 mg/kg, the plasma concentration reached peaks of 95.7, 219.9, 388.6 ng/ml at times of 1.22-1.78 h. The oral bioavailability was calculated as 15.6-20.3% in rat plasma. The result provided pre-clinical information for further application of aesculetin.