Determination of fraxetin, fraxin and dimethylfraxetin in rat plasma by UPLC-MS/MS

Abstract

The levels of fraxetin, fraxin, and dimethylfraxetin in rat plasma to be measured using an ultra-performance liquid chromatography tandem mass-spectrometry (UPLC–MS/MS) technique and applied to their pharmacokinetics and bioavailability.The protein precipitation technique was applied to the plasma preparation using acetonitrile and methanol (9:1, v/v). At a flow rate of 0.4 mL min−1, the elution time was 6 min. The mobile phase consisted of acetonitrile-water with 0.1% formic acid, and the chromatographic column was UPLC HSS T3 (50 mm × 2.1 mm, 1.8 μm). Quantitative analysis was conducted using multiple reaction monitoring (MRM) mode and detection was performed using electrospray ionization (ESI) positive ion mode. In each group, six rats were treated with fraxetin, fraxin, and dimethylfraxetin either orally (5 mg kg−1) or intravenously (1 mg kg−1).The calibration curves showed good linearity in the range of 2–4,000 ng mL−1, where r was greater than 0.99. The bioavailability of dimethylfraxetin, fraxin, and fraxetin was determinated to be 19.7, 1.4, and 6.0%.The established UPLC-MS/MS method for determining the levels of these three compounds in rat plasma was successfully applied to the pharmacokinetics of dimethylfraxetin, fraxin, and fraxetin, and the bioavailability was calculated.