2.6. LC–MS/MS analysis

To quantify cinnamic acid derivatives in the plasma, LC–MS/MS analysis was performed using a UHPLC system (Ultimate 3000, Thermo Scientific) coupled to an Orbitrap MS system (Q‐Exactive Focus, Thermo Scientific) according to a previously described method (Yamaga et al., ^{2021, 2022}). Briefly, 5 μL of the sample was injected into a reversed‐phase column (Acquity UPLC BEH C18, 2.1 × 100 mm, ID. 1.7 μm; Waters, Milford, MA, USA) held at 30°C. The elution was performed using solvent A (0.1% formic acid) and solvent B (acetonitrile) at a flow rate of 0.3 mL/min. The gradient profiles were as follows: 5% B (0–2 min), 5–95% B (2–22 min), 95% B (22–25 min), and 5% B (25–30 min). Tandem mass spectrometry (MS/MS) was conducted using an ion trap mass spectrometer with a heated electrospray ion source (HESI) in both positive and negative ionization modes (scan range m/z 70–1000). The mass detection parameters were the same as previously described (Tani et al., ²⁰²³). External standard curves for cinnamic acid derivatives were generated using eight concentrations of authentic standard solutions (1, 5, 10, 50, 100, 250, 500, and 1000 ng/mL).

The in vitro samples were analyzed using HPLC (LC‐20A, Shimadzu, Kyoto, Japan) with a reversed‐phase column (Inertsil ODS‐3, 2.1 × 33 mm, ID. 3 μm, GL Sciences, Tokyo, Japan) held at 40°C. The mobile solvents used were water with 10 mM ammonium acetate (pH 4.0, A) and methanol (B) pumped at a flow rate of 0.4 mL/min. The following gradient was employed: 10%–90% B (0–0.5 min), 90% B (0.5–2.5 min), and 10% B (2.5–4 min). The eluent was monitored using a triple quadrupole mass spectrometer (API 4000, AB Sciex, Framingham, MA, USA) incorporating an electrospray ion source (ESI) operating in the multiple reaction monitoring mode. The ESI source was operated in negative ionization mode at 600°C with a spray voltage of −4.5 kV. The curtain gas (nitrogen), collision gas (nitrogen), nebulizer gas (whole air), and drying gas (whole air) were set at 40, 5, 50, and 70 psi, respectively. The monitor ions of artepillin C and drupanin were m/z 299/255 [M − H]⁻ and m/z 231/132 [M − H]⁻, respectively. Artepillin C and drupanin were diluted in 50% methanol to concentrations of 1.6, 3.2, 8, 40, 200, 1000, and 2000 nM and were used as external standards. Sulfaphenazole (0.2 μM) was used as an internal standard.