## 2 Materials and Methods
### Characterization of the Lipid Profile of Red Raspberry Juice
#### 2.6.2 Analysis of the Lipid Metabolites  
The lipid metabolites of fresh, HHP-, and HTST-treated red raspberry juice were characterized and compared by an untargeted metabolomics strategy as described in our previous work [17]. The pre-frozen juice was freeze-dried in a lyophilizer for 48 h at a temperature of -40 \({}^{\circ}\)C \(\pm\) 2 \({}^{\circ}\)C and a vacuum pressure of <5.00 Pa (LG)-25C, Foring Technology Development Co., Ltd., Beijing, China). The freeze-dried sample was crushed using a mixer mill with a zirconia bead for 1.5 min at 30 Hz (MM 400, Retsch GmbH, Haan, Germany). Afterwards, 100 mg powder was mixed with 1.0 mL 70% aqueous methanol and extracted at 4 \({}^{\circ}\)C overnight. Following centrifugation at 10,000\(\times\)\(g\) for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 mL; ANPEL Laboratory Technologies Inc., Shanghai, China) and filtrated (SCAA-104, 0.22 um pore size; ANPEL Laboratory Technologies Inc.). The sample extracts were analyzed using an ultra-performance liquid chromatograph (UPLC)/electrospray ionization (ESI)-MS/MS system (UPLC, Shimadzu CBM30A, Shimadzu Corporation, Kyoto, Japan; MS, AB SCIEX QTRAP 6500, AB SCIEX Instruments, Framingham, MA, USA).  
The analytical conditions of UPLC were as follows: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 um, 2.1 mm \(\times\) 100 mm); solvent system, ultrapure water containing 0.04% (\(v/v\)) acetic acid as phase A, acetonitrile containing 0.1% (\(v/v\)) acetic acid as phase B; gradient program, time gradient: 0 min \(\rightarrow\) 11 min \(\rightarrow\) 12 min \(\rightarrow\) 15 min, concentration gradient (proportion of phase A): 100% \(\rightarrow\) 5% \(\rightarrow\) 5% \(\rightarrow\) 95%; flow rate, 0.40 mL/min; temperature, 40 \({}^{\circ}\)C; injection volume: 2 \(\upmu\)L [25].  
Linear ion trap (LIT) and triple quadrupole (QqQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature, 500 \({}^{\circ}\)C; ion spray voltage (IS), 5500 V; ion source gas I (GSI), gas II (GSI), and curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 umol/L polypropylene glycol solutions in QqQ and LIT modes, respectively. QqQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. The declustering potential (DP) and collision energy (CE) for individual MRM transitions were conducted with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the metabolites eluted within this period [26].  
Primary and secondary MS data, including the accurate precursor ion (Q1) and production (Q3) values, retention time (Rt), and fragmentation patterns, were subjected to qualitative analysis by comparing them to the data obtained from the self-built database MWDB (MetWare Biological Science and Technology Co., Ltd., Wuhan, China) and publicly available metabolite databases [27].