*4.5. LC-MS/MS Analysis of Free Amino Acids*

The amino acid composition and content of the OPs were measured, as previously described, with little modification [92]. The OPs samples were accurately weighed to 50 mg and mixed with 600 μL of a water–methanol solution (1:1, *v*/*v*) with 10% formic acid in a 2 mL tube. Then, 100 mg of glass beads were added to the mixed samples and vortexed for 30 s. The samples were transferred to a high-throughput tissue grinding machine (MB-96, Meibi, Jiaxing, Zhejiang, China) and vibrated at 60 Hz for 2 min. The tube was centrifuged at 12,000 rpm for 5 min at 4 ◦C. Ten μL of supernatant was transferred to a new tube containing 490 μL of the water–methanol solution (1:1, *v*/*v*) with 10% formic acid and then vortexed for 30 s. Then, 100 μL of the diluted samples were mixed with 100 μL of 100 μg/L double isotope internal standard (Trp-d3, D87103, Medical Isotopes, USA) and vortexed for 30 s. The mixed samples were filtered through a 0.22 μm hydrophilic PTFE filter and transferred into a labeled vial, and subsequently analyzed via LC–MS/MS.

Five μL of the samples were injected into an ACQUITY UPLC BEH C18 column (2.1 × 100 mm,1.7 μm, Waters, Milford, MD, USA) with mobile phase A: 10% water -methanol solution with 0.1% formic acid; B: 50% water-methanol solution with 0.1% formic acid. The flow rate: 300 μL/min in 8.5 min, then kept 300–400 μL/min for 4 min. The gradient elution programs: 0~6.5 min, 10~30% B; 6.5~7 min, 30~100% B; 7~8 min, 100% B; 8~8.5 min, 100~10% B; 8.5~12.5 min, 10% B.

Mass spectrometric analysis was performed using an AB SCIEX AB4000 Mass Spectrometer (AB SCIEX, Framingham, MD, USA) equipped with an electrospray ionization (ESI) source using the following parameters: capillary voltage: 5500 V, temperature of the turbo heaters: 500 ◦C, nebulizer gas (GS1): 50 psi, auxiliary gas (GS2): 50 psi, and curtain gas (CUR): 30 psi, Collision Gas: 6 psi. All of the amino acids were detected in the multiple reaction monitoring mode (MRM).
