Qtrap 6500 system

After 5 days of HA and β‐TCP treatment respectively, RAW264.7 cells were collected and centrifuged at 1200 r min⁻¹ for 5 min at 4 °C, before washing them in cold PBS. Further centrifugation at 1200 r min⁻¹ for 5 min at 4 °C was followed by vigorous vortexing with 1 mL of 80% cold methanol. The supernatant was collected and normalized to the protein concentration. Subsequently, the sample extracts were analyzed using an LC‐ESI‐MS/MS system (Waters ACQUITY H‐Class; MS, QTRAP 6500+ System). The AB 6500+ QTRAP LC‐MS/MS system was equipped with an ESI Turbo Ion‐Spray interface and can operate in both positive and negative ion modes according to Analyst 1.6 software (A B Sciex). Detection and determination of the relative abundances of the targeted energy metabolites were performed using MetWare based on the AB Sciex QTRAP 655 LC‐MS/MS platform.

The experimental protocols were validated in previous studies [32 ]. Heart tissues were stored at − 80 °C, thawed in a centrifuge tube, and ground away from light. Methanol (0.2 mL) containing internal standard was added to the 20 mg heart sample to precipitate proteins. Butylated hydroxytoluene (0.005%) were added into solution to avoid oxidative reaction during extraction. The eicosanoids in supernatants were extracted using Poly-Sery MAX SPE columns (ANPEL). The extraction was analyzed using a LC-ESI-MS/MS system (UPLC, ExionLC AD; MS, QTRAP®6500 + System; Column, Waters ACQUITY UPLC HSS T3 C18 100 mm × 2.1 mm, i.d. 1.8 μm). The binary gradient consisted of (A) 0.04% water/acetic acid and (B) 0.04% acetonitrile/acetic acid for 10 min. Metabolic features of oxylipins were analyzed using scheduled multiple reaction monitoring (MRM). The contents of oxylipins in heart tissues were quantified according to the calibration curve for each eicosanoid (Table S3).

The sample extracts were analyzed using an LC-ESI-MS/MS system (UPLC, ExionLC¹; MS, QTRAP® 6,500+ System).² The analytical conditions were as follows: T3 method˖HPLC: column (Waters ACQUITY UPLC HSS T3 C18; 100 mm × 2.1 mm i.d.ˈ1.8 μm); solvent system, water with 0.05% formic acid (A); ACN with 0.05% formic acid (B). The gradient was initiated at 5% B (0 min), increased to 95% B (8–9.5 min), and finally ramped back to 5% B (9.6–12 min); flow rate, 0.35 mL/min; temperature, 40°C; injection volume: 2 μL.
Amide method: HPLC column, ACQUITY UPLC BEH Amide (i.d.2.1 × 100 mm, 1.7 μm); solvent system, water with 10 mM ammonium acetate and 0.3% ammonium hydroxide (A), 90% ACN/water (V/V) (B); The gradient was initiated at 95% B (0–1.2 min), decreased to 70% B (8 min), 50% B (9–11 min), and finally ramped back to 95% B (11.1–15 min); flow rate, 0.4 mL/min; temperature, 40°C; injection volume: 2 μL.