2.8.3. Sample Preparation

The method of sample preparation was a modified method of Isleyen et al. [34] for determination of fexofenadine plasma concentration. In summary, 50 μL of 50-ng/mL terfenadine in acetonitrile solution was added to a 50-μL aliquot of plasma, then 20 μL of aqueous 13-μM formic acid solution was added. After vortexing, 50 μL of extraction solvent (a mixture of dichloromethane, ethyl acetate, diethyl ether at the ratio of 30:40:30, *v*/*v*/*v*) was added. The sample was then vortexed for 40 s. The protein precipitation was performed via centrifugation at 10,000× *g* and 4 ◦C for 5 min. The supernatant was cooled at −80 ◦C for 10 min. The upper fraction of the supernatant was transferred to a polypropylene tube and evaporated with N2 gas. After being reconstituted with 200 μL of the mobile phase initial composition [5-mM ammonium formate (at a pH of 4): acetonitrile = 60:40], an aliquot of 2 μL was injected into LC-MS/MS.

#### *2.9. Physicochemical Interaction Study*

To investigate the possible physicochemical interactions between drug and *R. acetosa* extract, Fourier transform infrared (FT-IR) spectrum measurement and solubility test were carried out.

FT-IR spectra of fexofenadine HCl, *R. acetosa* extract and mixture of fexofenadine-extract (1:1) were measured by Nicolet iS 50 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA) with attenuated total reflectance (ATR) mode.

The change on the solubility of fexofenadine after mixing with *R. acetosa* extract was tested. The method was modified previously reported method [35,36]. Briefly, 200 μg of fexofenadine and *R. acetosa* extract were placed in the tube after centrifugal vacuum evaporation of solvent. The control group has fexofenadine only, and the mixed group has both fexofenadine and the extract. A 200-μL aliquot of the simulated intestinal fluid (SIF, pH 6.8) without enzyme [37] was added to each tube. The tubes were then incubated in a shaking water bath at 37 ◦C for 12 h. The concentration of fexofenadine was 1 mg/mL, corresponding to the orally administered concentration to the rats (10 mg/5 mL/kg-fexofenadine with 5-mL/kg extract, total 10 mL). After the incubation, the tubes were centrifuged at 10,000× *g* for 10 min. The supernatant was filtered, diluted with mobile phase, and analyzed by LC-MS/MS.
