metabolites relative contents of all samples

erwent independent analysis with three biological replicates. The data acquisition system primarily comprised UPLC (SHIMADZU Nexera X2) coupled with Tandem mass spectrometry (Applied Biosystems 4500 QTRAP). The liquid phase conditions predominantly featured Agilent SB-C18 (1.8 μm, 2.1 mm*100 mm), with the solvent system comprising ultrapure water (0.1% formic acid, referred to as solvent A) and acetonitrile (0.1% formic acid, referred to as solvent B). The elution gradient commenced at 95:5 for A/B at 0 minutes, transitioned to 5:95 for A/B at 9 minutes, reverted to 95:5 for A/B at 10-11 minutes, and maintained 95:5 for A/B at 14 minutes. The flow rate was set at 0.35 mL/min, the column temperature maintained at 40°C, and the injection volume set at 4 μL.The mass spectrometry conditions primarily encompassed LIT and triple quadrupole (QQQ) scans conducted on a state-of-art triple quadrupole linear ion trap mass spectrometer (Q TRAP), specifically the AB4500 Q TRAP UPLC-MS/MS system equipped with an ESI Turbo ion spray interface. This system is adeptly controlled by the Analyst 1.6.3 software (AB Sciex), allowing operation in positive and negative ionization modes. The ESI source was configured with a source temperature of 550°C, an ion spray voltage of 5500 V (in positive ion mode) and -4500 V (in negative ion mode), while the ion source gas I, gas II, and curtain gas were set to 50, 60, and 25 psi, respectively. The QQQ scans were executed in MRM mode, with DP and CE parameters meticulously calibrated for each MRM ion pair. Tailored to the eluted metabolites in each period, a distinct set of MRM ion pairs was diligently monitored.