Lc 30ad system

Cells were lysed in 1 mL TCEP solution and then incubated for 10 min at laboratory temperature and sonicated for 2 × 1 min in a pulse mode. 200 μL of cell lysate was transferred to a vial containing 800 μL methanol + 1% FA and vortexed for 10 s and then stored at −20°C for 2 h. The sample was centrifuged at 13000 rpm for 10 min at 4°C and the supernatants were pipetted and filtered through a 0.22-μm filter and placed into vials for LC-MS/MS analysis. The LC-MS/MS analysis was performed using a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an LC-30AD system (Shimadzu, Kyoto, Japan) and SIL-30AD autosampler (Shimadzu, Kyoto, Japan). Chromatographic separation was performed using gradient elution on a reversed-phase UPLC XSelect HSS T3 1.8 μm, 100 × 2.1 mm I.D. column (Waters, Milford, MA, USA). The mobile phases were as follows: A, 10 mM ammonium acetate in water and B, 20% (v/v) acetonitrile in methanol. The flow rate was 0.3 mL/min. The gradient employed was as follows: 0.01–1.5 min started at 5% B, 1.5–6 min from 5% to 50% B, 6–6.1 min from 50% to 95% B and hold for 2 min, and 8–9 min from 95% to 5% B and hold for 4 min. The total time was 13 min. The sample temperature in the autosampler was maintained at 4°C, and the injection volume was 1 μL in each run.

The plasma concentration of each drug was measured by LC-MS/MS based on the ESI (electrospray ionization) method. The LC-MS/MS system consisted of a high-performance liquid chromatograph LC-30AD system (Shimadzu, Kyoto, Japan) and an AB Sciex QTRAP 6500 tandem mass spectrometer (Framingham, MA, USA). Capcell Pak C18 MGII (particle size 5 μm, inner diameter 2.0 mm x 50 mm; Osaka Soda, Osaka, Japan) was used as the separation column. To measure the concentration of SVA, solvent A (water containing 0.063% ammonium formate) and solvent B (acetonitrile) were used as the mobile phase. The gradient conditions were maintained at 20% solvent B for 1.5 min, increased from 20% to 95% solvent B in 1.50 min, held at 95% for 1.0 min, decreased back to 20% within 0.01 min, and held at 20% for 1.49 min. To measure the concentration of LEV and CBZ, solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile) were used as the mobile phase. The gradient conditions of LEV were increased from 5% to 60% solvent B in 1.50 min, sharply increased to 95% within 0.01 min, held at 95% for 1.49 min, decreased back to 5% within 0.01 min, and held at 5% for 1.49 min. The gradient conditions of CBZ were increased from 30% to 95% solvent B in 2.00 min, held at 95% for 1.00 min, decreased back to 30% within 0.01 min, and held at 30% for 1.49 min.

Anthocyanins were determined by LC-MS/MS analysis using a Shimadzu LC 30AD system combined with a Shimadzu 8030+ MS detector with an electrospray ionization (ESI) source. The separation of anthocyanins was made at 40 °C with a Zorbax Eclipse Plus C18—2.1 mm × 50 mm–1.8 µm column (Agilent Technology, Santa Clara, CA, USA). The mobile phase consisted of aqueous 0.5% formic acid (A) and methanol 0.5% formic acid (B). The flow rate was set at 0.5 mL/min. The samples were eluted using a linear gradient: 0–1 min, 15% B; 1–5 min, 15–50% B; 5–5.1 min, 50–100% B; 5.1–6.0 min, 100% isocratic; 6.0–6.1 min, 100–15% B. The sample injection volume was set at 5 µL. All the MS/MS parameters were optimized by the infusion of each of the standards (1 µg/mL) and the optimal ionization conditions and fragmentation patterns were determined using instrument software (Labsolutions, Shimadzu, Kyoto, Japan). Multiple reaction monitoring (MRM) parameters for each anthocyanin standard are presented in Table 1. In the positive ion mode, the mass spectrometry conditions were as follow: nebulizer gas flow, 2 L/min; drying gas flow, 10 L/min; heating block temperature 400 °C; DL temperature 250 °C; and mass range from 50 to 1200 m/z. Standard calibration curves were constructed for all analytes and the concentration of anthocyanins in the samples was determined using regression analysis (R² > 0.99).

Analyses were performed on LS-MS/MS analyses with a Shimadzu LC-30AD system coupled with a Shimadzu MS-8060 mass spectrometer (Kyoto, Japan). ZORBAX SB-C18 column (50 mm × 2.1 mm × 1.8 µm) was purchased from Agilent (Santa Clara, CA, USA). CPA224S electronic analytical balance was purchased from Sartorius Company (Göttingen, Germany). The refrigerator was obtained from Siemens (Munich, Germany). The HITACHI CT6E centrifuge was purchased from Hitachi, Ltd. (Tokyo, Japan). The 80350-CN grinder was supplied by Hamilton Beach Electric Co., Ltd. (Shenzhen, China). A DFT-100A portable high-speed universal crusher was purchased from Linda Machinery Co., Ltd. (Wenling, China). The HQ-60 vortex mixer was purchased from North Tongzheng Biotechnology Co., Ltd. (Beijing, China). The DTC-27J ultrasonic cleaner was provided by Dingtai Biochemical Technology Equipment Manufacturing Co., Ltd. (Wuhan, China). SPE column (Mega BE-SI, 1 g, 6 mL) was purchased from Agilent (Santa Clara, CA, USA). Pipettes (10 µL, 200 µL, 1 mL, 5 mL) were purchased from Eppendorf Company (Hamburg, Germany). A 1 mL syringe was purchased from Wangguan Medical Devices Co., Ltd. (Wuhan, China). A 0.22 µm organic phase filter was purchased in Millipore Company (Burlington, MA, USA).