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and the extraction recovery and matrix effect met the requirements of the determination. Therefore, caffeine was selected as the internal standard.

#### *2.2. Method Validation*

#### 2.2.1. Selectivity and Carry-Over

No interference peaks were observed at the retention time of alnustone and IS in the chromatograms of six batches of the blank plasma samples. Figure 2 illustrated representative chromatograms of the blank samples, the blank samples spiked with alnustone at the lower limit of the quantification (LLOQ) and IS, and the samples after alnustone administration. The response of the blank samples was compared with the LLOQ samples. The results suggested that there was no obvious endogenous interference in the determination of alnustone and IS. *Molecules* **ŘŖŗş**ǰȱ*24*ǰȱ¡ȱȱȱȱ śȱȱŗŝȱ

**ȱŘǯȱ**ȱȱDZȱǻǼȱȱǻśǯŞŚȱǼȱȱȱǻřǯřřȱǼȱȱ¢ȱ ȱ¡ȱȱȱDzȱǻǼȱȱȱȱ ȱȱȱDzȱǻǼȱȱȱ ȱȱȱŘȱȱȱȱȱȱȱǻśȱȦǼȱȱDzȱǻǼȱȱȱȱ ȱȱ ȱȱȱDzȱǻǼȱȱȱȱ ȱȱȱǯȱ **Figure 2.** Representative chromatograms of: (**A**) alnustone (5.84 min) and IS (3.33 min) obtained by the extraction of blank plasma; (**B**) blank plasma spiked with alnustone and IS; (**C**) plasma sample from a rat 2h after an intravenous administration of alnustone (5 mg/kg) to rats; (**D**) blank liver tissue homogenates spiked with alnustone at LLOQ; (**E**) blank plasma spiked with alnustone at LLOQ.
