## 4 Materials and Methods
### Analysis of Nonvolatile Compounds by HPLC-MS
#### 4.4.2 Detection of Non-VOCs  
The extracted samples were subjected to analysis using an UPLC-ESI-MS/MS system comprising a Shimadzu 30A UPLC (Kyoto, Japan) and an Applied Biosystems 5600 Q-TOF/6500 Q-TRAP Mass Spectrometer (AB sciex, Boston, MA, USA). Liquid chromatography tandem time-of-flight mass spectrometry (5600 Q-TOF) was utilized to determine the accurate molecular weights of each metabolite's standard. It provided detailed measurements of the different peak integrations and secondary fragmentation information, which were crucial for identifying and quantifying the substances. Liquid chromatography tandem quadrupole mass spectrometry (6500 Q-TRAP) operates based on the standard metabolite information, employing multiple reaction monitoring (MRM) to conduct simultaneous detection. This method enhances the precision and reliability of the quantification processes by targeting specific metabolite transitions, thereby ensuring high specificity in detection. The detection and analysis methods were optimized following the protocol previously described by [PERSON] et al. (2016) and carried out by Verygenome Technology Co., Ltd. (Guangzhou, China). Both instruments utilized the same chromatographic conditions and electrospray ionization (ESI) source.  
Chromographic conditions: A Waters UPLC BEH Amide column (1.7 um, 2.1 \(\times\) 100 mm) was used to analyze the non-VOCs. Solvent A comprised ultrapure water with 25 mM CH\({}_{3}\)COONH\({}_{4}\) and 25 mM NH\({}_{4}\)OH, and solvent B comprised chromatographically pure acetonitrile. The gradient elution procedure was as follows: starting at 0 minutes with 90% solvent B; from 0 to 9 min, the proportion of solvent B is reduced to 40%; at 9 to 9.05 minutes, it returns to 90% B; and maintains 90% B from 9.05 to 11.5 min. The flow rate was 0.3 mL/min. The injection volume was 5 um, and the column temperature was 55 \({}^{\circ}\)C. The effluent was alternatively directed to the ESI-Q-TOF-MS system.  
Mass spectrum conditions: The ESI source operation parameters were as follows: the source temperature was 600 \({}^{\circ}\)C; the ion spray voltage was 5500 V in positive ion mode and \(-\)4500 V in negative ion mode; the curtain gas pressure was 20 psi; and the nebulizer and auxiliary gas pressure was 60 psi. Scanning was performed via multiple reaction monitoring (MRM). With the collision gas (nitrogen) adjusted to the medium during the multiple reaction monitoring mode (MRM) tests, QQQ scans were collected. The raw data were converted to the '.mxXML' format via ProteoWizard, and peak alignment, retention time correction, and peak area extraction were performed via the XCMS program ([[http://enigma.lbl.gov/xcms-online/](http://enigma.lbl.gov/xcms-online/)]([http://enigma.lbl.gov/xcms-online/](http://enigma.lbl.gov/xcms-online/)), accessed on 17 September 2024). Metabolite structures were accurately matched (<25 ppm) using primary and secondary spectra obtained from a self-constructed database (VGDB) and public databases. The data were normalized, statistically analyzed and then plotted via R software (version R-4.3.2).  
Quantitative and qualitative analyses of non-VOCs: To accurately identify the metabolites, the retention time, fragmentation pattern, and precise _m/z_ value were compared to standards in the self-compiled metabolite database (VGTB, Guangzhou, China). The quantitative analysis was conducted based on the signal intensity of metabolites derived from various ions. MS signals, including isotopes, K\({}^{+}\), Na\({}^{+}\), and NH\({}_{4}\)\({}^{+}\) adduct ions, were eliminated. Chromatographic peaks were meticulously merged and modified with the MultiQuant software (version 3.0.3). The area beneath each chromatographic peak indicated the relative concentration of the relevant chemical.  
Analysis of Volatile Compounds by Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry (SPME-GC-MS)