2.3.3. Identification of OCT2 808G>T

Candidate SNP (808G>T) in the *OCT2* gene was genotyped using pyrosequencing analysis. Each primer was prepared according to the variation of allele. Forward primer of 5′ -CGGAGAACAGT GGGGATTTTTTAC-3′ , reverse primer of 5′ -CACGTAATTCCTTCCGTCTGAAGA-3′ , and sequencing primer of 5′ -GGTGGTTGCAGTTCACA-3′ were used for *OCT2* 808G>T genotype. Forward primer has a 5′ biotin triethylene glycol label necessary for post PCR processing. PCR was performed in 20 µL reaction mixture including 1 µL extracted DNA, 1 µL of 10 pmol each primer (forward and reverse), and 17 µL autoclaved distilled water. The PCR program comprised an initial denaturation at 94 ◦C for 5 min followed by 45 cycles of denaturation at 94 ◦C for 20 s, annealing at 52.1 ◦C for 30 s, and extension at 72 ◦C for 20 s. A final extension step was performed at 72 ◦C for 5 min. The biotinylated PCR products were immobilized on streptavidin-coated Sepharose beads (Amersham Biosciences, Uppsala, Sweden). A total of 37 µL of binding buffer (10 mM Tris HCl, 2 M sodium chloride, 1 mM EDTA, 0.1% Tween 20, pH 7.6; Pyrosequencing AB, Uppsala, Sweden), 3 µL of streptavidin-coated Sepharose beads, and 20 µL of water were added to 20 µL PCR product; then, the solution was vigorously shaken for 10 min at room temperature. A 96 pin magnetic tool (Pyrosequencing AB, Uppsala, Sweden) was used to transfer up

to 96 samples at a time to solutions as follows. The beads with bound template were first transferred to 70% ethanol solution and 0.2 N sodium hydroxide solution, then to 1X washing buffer (Pyrosequencing AB, Uppsala, Sweden), and finally into a solution of 1X annealing buffer (20 mM Tris acetate, 2 mM magnesium acetate, pH 7.6), including the appropriate sequencing primer of 10 pmol. Lastly, this mixture was heated for 1 min to 95 ◦C and then cooled to 50 ◦C and incubated at room temperature for at least 5 min to bind the sequencing primer to the template. After template preparation, a 96-well plate including the samples was loaded into the instrument (PSQ 96MA; Pyrosequencing AB, Uppsala, Sweden) along with the optimal reagents (Pyro Gold; Biotage AB, Uppsala, Sweden). This instrument sequences the templates by dispensing reagents and deoxynucleotide triphosphates in a user-defined order, achieving real-time sequencing by synthesis in an automated fashion. This is achieved by creating and monitoring an enzyme cascade initiated by nucleotide incorporation that produces light emission. Pyrosequencing data were obtained by using Peak Height Determination Software v2.1 (Pyrosequencing AB, Uppsala, Sweden).

#### 2.3.4. Identification of PEPT1 1287G>C (in Exon 16)

Candidate SNP (1287G>C) in the *PEPT1* gene were genotyped by using PCR-RFLP. Each primer was prepared according to the variation of allele. Forward primer of 5′ -CCCTTGTCAGGGTTAAGATGA-3′ and reverse primer of 5′ -GCTTCTCTAAATCCTATTATAACAGGG-3′ were used for *PEPT1* 1287G>C genotypes. PCR was performed in 20 µL reaction mixture including 1 µL extracted DNA, 1 µL of 10 pmol each primer (forward and reverse), and 17 µL autoclaved distilled water. The PCR program comprised of an initial denaturation at 95 ◦C for 5 min followed by 35 cycles of denaturation at 95 ◦C for 20 s, annealing for at 54.5 ◦C 30 s, and an extension at 72 ◦C for 20 s. The final extension step was performed at 72 ◦C for 5 min. DNA fragments amplified by PCR were reacted at 37 ◦C for 1 h with restriction enzyme *Sau961*, which can recognize and cleave specific sequences. The digested fragments were separated by electrophoresis in 2.5% agarose gel, and were visualized under ultraviolet light after staining the gel with ethidium bromide for 30 min.

#### *2.4. Determination of Plasma Tiropramide Concentrations*

Plasma concentrations of tiropramide were determined using a validated column-switching semi-micro high-performance liquid chromatography (HPLC) method based on a previous study [12].

#### 2.4.1. Chromatographic Conditions

The analytical system consisted of the Nanospace SI-2 series (Shiseido, Tokyo, Japan) with an ultraviolet-visible (UV–VIS) detector 3002, two 3001 pumps, a 3014 column oven, a high pressure six-way switching valve 3012, a 3010 degasser, and a 3023 autosampler. The system operation and signal processing were operated by Syscon (Shiseido, Tokyo, Japan). The columns used in this on-line extraction system include an analytical column (Capcell Pak C<sup>18</sup> UG120, 150 × 1.5 mm I.D. Shiseido), a pre-column (Capcell Pak MF Ph-1, 10 × 4 mm I.D. 5 µm Shiseido), and an enrichment column (Capcell Pak C<sup>18</sup> UG120, 35 × 2 mm I.D. Shiseido). The mobile phase for primary separation of tiropramide in the pre-column and concentration in the enrichment column was phosphate buffer (50 mM, pH 7.0)-acetonitrile (88/12, v/v) with a flow rate of 0.5 mL/min. The mobile phase for analytical column was phosphoric acid–phosphate buffer (50 mM, pH 7.0)-acetonitrile (0.04/59.96/40, v/v/v) with a flow rate of 0.1 mL/min. All the columns were maintained at 25–30 ◦C. The quantification was performed at 230 nm wavelength. The peak with the retention time of tiropramide was verified using a photodiode array detector (2017 Diode Array, Shiseido, Tokyo, Japan).

#### 2.4.2. Analytical Procedures

The performance of column-switching semi-micro HPLC consists of three steps, as follows: sample loading and primary separation, enrichment of analyte fraction, and chromatographic separation. When the column-switching valve was at the precolumn inlet position, an aliquot of filtered (by 0.22 µm, Millex-GV syringe filter unit, Millipore, Burlington, MA, USA) plasma sample (80 µL) was loaded to the pre-column, and a primary separation of tiropramide from plasma proteins was conducted by using phosphate buffer (50 mM, pH 7.0)-acetonitrile (88/12, v/v). Subsequently, the valve was switched to the enrichment column inlet position, and the tiropramide fraction was eluted from the pre-column and concentrated in the enrichment column by phosphate buffer (50 mM, pH 7.0)-acetonitrile (88/12, v/v). Afterwards, the valve was switched to analytical column inlet position, and tiropramide was finally isolated and quantified by phosphoric acid–phosphate buffer (50 mM, pH 7.0)-acetonitrile (0.04/59.96/40, v/v/v).
