*2.5. Targeted Quantification of ECG and L-Theanine in CBP*

The contents of ECG and L-theanine in CBP were determined using the UHPLC system (Dionex Ultimate 3000, Thermo Scientific, Waltham, CA, USA) coupled with Q-Exactive Orbitrap-MS (Thermo Scientific, Waltham, CA, USA) in parallel reaction monitoring (PRM) mode. Compound separation was performed using hydrophilic interaction liquid chromatography (HILIC) with an ACQUITY BEH Amide column (150 mm × 2.1 mm, 1.7 μm, Waters, Milford, MA, USA) at 50 ◦C. Mobile phase A and B were 30% and 95% acetonitrile, respectively, both containing 10 mmol/L ammonium formate and 0.1% formic acid. A gradient elution profile at a flow rate of 0.3 mL/min was set as follows: 1 min, 100% B; 11 min, 30% B; 11.5 min, 100% B; and 15 min, 100% B. The injection volume was 2 μL. The Q-Exactive Orbitrap-MS with a heated HESI source was operated in negative mode with stepped normalized collision energies (NCE 10, 20, and 30). The mass resolution was 70,000 full width at half maximum (FWHM) for the full MS mode and 17,500 FWHM for the MS/MS scan. Calibration curves were established with standard solutions (0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μg/mL for ECG and 5, 10, 20, 40, 60, 80, and 100 μg/mL for L-theanine) and used to calculate the ECG and L-theanine content in Xcalibur (v4.0.27.19, Thermo Fisher Scientific, Waltham, MA, USA).
