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For the first time, a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of fraxin and its metabolite, fraxetin, in rat plasma, using esculin as the internal standard (IS). The plasma samples were precipitated with methanol before separation on an Nova-Pak C 18 column (150 mm × 3.9 mm, 3 μm) using a mobile phase consisting of 0.1% formic acid and methanol (55:45) at a flow rate of 0.8 mL/min. The analytes were detected by multiple reaction monitoring in the negative ion mode with the mass transitions at m / z 368.9→ m / z 191.9 (fraxin), m / z 206.9→ m / z 191.8 (fraxetin) and m / z 339.0→ m / z 176.9 (esculin, IS). The results demonstrated that the calibration curves for both analytes have good linearity (r ≥ 0.995) over a concentration range of 5.00–3000 ng/mL. The assay was validated according to the regulatory bioanalytical guidelines and proved acceptable. The intra- and inter-day precisions (R.S.D.%) were within 10.9% for both analytes, whereas the deviation of assay accuracies (R.E.%) ranged from −5.3 to 1.0%. The method was successfully applied to a pharmacokinetic study after a single oral dose of fraxin at 50 mg/kg to rats. [ABSTRACT FROM AUTHOR]