Lc 30a

The concentrations of analytes were determined by UPLC–MS/MS, which consisted of an LC-30A ultra-performance liquid chromatography (Shimadzu, Japan), and Sciex Triple Quad 5500 tandem triple quadrupole mass spectrometer (Applied Biosystems Sciex, Framingham, MA, USA). The XSelect HSS T3 column (2.1 mm × 100 mm, 2.5 μm, Waters, Milford, MA, USA) was used for chromatographic separation. Phase A (0.1% formic acid in ultrapure water containing 5 mM ammonium acetate) and phase B (acetonitrile) were used as the mobile phases for gradient elution at 0.35 mL/min. The following gradient conditions for phase B were used: 0–1 min, 60% B; 1–2 min, 60–90% B; 2–4 min, 90% B; 4.0–4.1 min, 90–60% B; and 4.1–5.1 min, 60% B. The positive ion mode with multi-reaction detection was used, and the multiple reaction monitoring transitions of the analytes were as follows: m/z 465.2→270.2 for sorafenib, 468.2→255.4 for sorafenib-d₃, m/z 427.0→370.0 for lenvatinib, 432.1→370.0 for ²H₅-lenvatinib, 462.1→191.3 for canagliflozin, and 466.1→195.3 for ²H₄-canagliflozin.

For liquid chromatography–mass spectrometry (LC-MS), 200 μL of each sample was mixed with 800 μL of precooled methanol/acetonitrile (1:1) for the precipitation of the proteins. The mixtures were centrifuged at 15,000xg for 4 min at 4°C. Then, the supernatants were collected and dried under vacuum. A solution containing acetonitrile and water (1:1; 100 μL) was added. The resulting solution was centrifuged at 14,000xg for 15 min at 4°C. The treated supernatants were analyzed using a liquid chromatograph LC-30A (SHIMADZU, Japan) and 4500 QQQ mass spectrometer (AB Sciex, USA).

Chromatographic analysis was conducted on an LC-30A ultra-high-performance liquid chromatograph (Shimadzu, Japan), equipped with an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) and a BEH C18 VanGuard pre-column (2.1 mm × 5 mm, 1.7 µm). The mobile phase consisted of 0.2% formic acid in H₂O (A) and acetonitrile (B). The gradient elution procedure was as follows: 0–2 min, 4%–6% B; 2–5 min, 6%–8.5% B; 5–6 min, 8.5%–12% B; 6–8 min, 12%–15% B; 8–14 min, 15%–18% B; 14–16 min, 18%–24% B; 16–18 min, 24%–100% B; 18–19 min, 100%–4% B; and 19–22 min, 4% B. The mobile phase operated at a flow rate of 0.25 mL/min, and the injection volume was 2 μL. The column temperature was maintained at 40°C.
All analytes were detected by a Q-TRAP™ 4500 MS/MS system (AB Sciex, United States) equipped with an electrospray ionization (ESI) source for mass spectrometry detection. Compound-dependent MS parameters, including precursor ion (Q1), production (Q3), declustering potential (DP), and collision energy (CE) were designed and optimized using the multiple reaction monitoring (MRM) modes. Other MS parameters were set as follows: source temperature, 500°C; ion source gas 1, 40 psi; ion source gas 2, 40 psi; curtain gas, 40 psi; and dwell time, 50 ms. Data was acquired and processed using AB Sciex Analyst 1.5.2 software (AB Sciex, United States).

The free amino acids (FAAs) were quantified using ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The FAA detection referred to another method with some modifications [61 (link)]. The initial amount of leaf tissue for each sample homogenate was 80 mgFW. Ultra-performance liquid chromatography (UPLC) was applied by using the chromatograph LC-30A (Shimadzu, Kyoto, Japan), mass spectrometer QTRAP 5500 (AB SCIEX, Framingham, MA, USA), and ACQUITY UPLC BEH Amide column (particle size, 1.7 µm; 2.1 mm i.d.; 50 mm long; Waters, Milford, MA, USA). A total of 27 free amino acids were detected using this method (Table S4).