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The validated method was successfully applied to pharmacokinetic study in rats after intravenous (5 mg/kg) and oral (10, 25, 50 mg/kg) administration of 5-hydroxy-4-methoxycanthin-6-one. The result indicated that 5-hydroxy-4-methoxycanthin-6-one was quickly absorbed into the blood and reached the highest concentration at ~33.0-42.0 min, with moderate elimination half-life (0.85-2.11 h) and low bioavailability (16.62-24.42%) after oral administration. The study provided valuable information that can be used as a reference for studying other canthinone alkalo