# Analysis of the Changes in Volatile Components During the Processing of Enshi Yulu Tea
## 2 Materials and Methods
### Volatile Component Analysis  
Volatile components were extracted using a Head Space Solid Phase Microextraction (HS-SPME) method. The sample was removed from the \(-20\)\({}^{\circ}\)C refrigerator and transferred to a drying dish. After reaching room temperature, approximately 0.5 g of the sample was weighed and placed into a 20 mL headspace bottle, to which 6 mL of saturated NaCl solution and 10 \(\mu\)L of hexan-3-one (50 \(\mu\)g/mL) as an internal standard. At constant temperature (60 \({}^{\circ}\)C), after oscillating for 5 min, the DVB/CWR/PDMS extraction head was inserted for 15 min to absorb volatile components. After extraction, desorption at 250 \({}^{\circ}\)C for 5 min was performed before performing Gas Chromatography-Mass Spectrometry (GC-MS) analysis on each sample five times. To ensure instrument stability, for every 10 samples to monitor the repeatability of the analytical process, quality control samples were tested three times (Figures S1 and S2).  
GC-MS analysis was carried out using a GC system (8890, Agilent Technologies, Santa Clara, CA, USA) coupled with an MS detector (7000D, Agilent Technologies, Santa Clara, CA, USA). GC-MS chromatographic conditions included: DB-5 MS capillary column (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\mu\)m, Agilent J&W Scientific, Folsom, CA, USA), high purity helium carrier gas (purity not less than 99.999%) at 1.2 mL/min constant flow rate; inlet temperature 250 \({}^{\circ}\)C, without split injection, with solvent delay set at 3.5 min; programmed temperature rise starting at 40 \({}^{\circ}\)C for 3.5 min followed by an increase of 10 \({}^{\circ}\)C/min to 100 \({}^{\circ}\)C, 7 \({}^{\circ}\)C/min to 180 \({}^{\circ}\)C, and 25 \({}^{\circ}\)C/min to 280 \({}^{\circ}\)C, with a hold time of 5 min. GC-MS mass spectrometry conditions included: electron bombardment ion source (EI), ion source temperature (230 \({}^{\circ}\)C), quadrupole bar temperature (150 \({}^{\circ}\)C), mass spectrum interface temperature (280 \({}^{\circ}\)C), electron energy (\(-70\)eV); scanning mode selected ion detection mode (SIM); and qualitative and quantitative ion precision scanning (GB 23200.8--2016).  
The analysis of volatile compounds was performed with the assistance of MetWare ([[http://www.metware.cn/](http://www.metware.cn/)]([http://www.metware.cn/](http://www.metware.cn/)), accessed on 23 September 2022) corporation (Wuhan, China), a professional institution for analysis. The volatile compounds were characterized by matching their mass spectra against the data system library (MetWare Gas Chromatography, MWGC) and linear retention index. For each compound, one quantitative ion and two or three qualitative ions were carefully chosen. Each group of ions was sequentially detected during their respective time segments based on their elution order. To confirm the identification, the observed retention time was compared with the standard reference, and the presence of all selected ions in the sample spectrum after background subtraction was verified [16]. For improved quantitative accuracy, specific quantitative ions were chosen for integration and calibration. The internal standard, hexan-3-one (10 \(\mu\)L, 50 \(\mu\)g/mL), was employed, and the relative content of each volatile was computed as follows:  
\[C_{j}=\frac{A_{j}\times V_{i}\times C_{i}}{A_{i}\times m_{s}}\]  
where \(C_{j}\) denotes the mass concentration of each compound (\(\mu\)g/kg); \(A_{j}\) and \(A_{i}\) represent the chromatographic peak areas of the compound and internal standard, respectively; \(V_{i}\) means the volume of internal standard (10 \(\mu\)L); \(C_{i}\) means the concentration of the internal standard (50 \(\mu\)g/mL); \(m_{s}\) means the mass of sample powder (kg).