Quantitative analysis of 5-FU and its bacterial-derived metabolites by untargeted metabolomics

We incubated 5-FU (40 μM) with <i>C. freundii</i> strain (4×10⁸ CFU/mL) in 600 μL of NB medium for 24 h (n=3). A blank control group was included. Post-incubation, reactions were stopped with 600 µL ice-cold acetonitrile. After vortexing, 200 µL aliquots were mixed with an equal volume of acetonitrile and precipitated at -20°C for 1 h. Samples were then revortexed, centrifuged (12,500 rpm, 15 min, 4°C), and 100 µL of supernatant was collected for quantitative analysis using an ultraperformance liquid chromatography (UPLC)-Orbitrap-Exploris-120-MS/MS apparatus with a Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). The mobile phase consisted of NH₄HCO₃ (6.5 mM) (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The gradient elution program was established: 0–1 min, 2% B; 1–3 min, 2–60% B; 3–5 min, 60% B; 5–7 min, 60–100% B; 7–8 min, 100% B; 8–8.1 min, 100–2% B; and 10 min, 2% B. Samples (10 μL of injection volume) were separated and analyzed with the column temperature at 40°C. Data acquisition was performed using Xcalibur software.Mass spectrometry analysis employed full-scan (60,000 resolution, 60–900 <i>m/z </i>range) and data-dependent MS² (dd-MS²; 15,000 resolution) modes. Ion source parameters included: 2500 V spray voltage (negative mode), sheath/aux gases at 50/10 arb, and 300°C transfer tube temperature. Full-MS used 100 ms maximum injection time with standard AGC target; dd-MS² used auto injection time and stepped HCD collision energy (20, 40, 60%). Retention times for 5-FU and FBAL were 0.666 and 0.687 min, respectively. Data processing utilized MS-DIAL (ppm calculation), Sirius (validation), and MZmine (MS² export).

将5-氟尿嘧啶（5-FU，40 μM）与弗氏柠檬酸杆菌（C. freundii）菌株（4×10^8 菌落形成单位/毫升，CFU/mL）置于600 μL营养肉汤（NB）培养基中共孵育24小时，设置3次生物学重复（n=3）并增设空白对照组。孵育结束后，加入600 μL冰乙腈终止反应。涡旋混匀后，取200 μL等分试样与等体积乙腈混合，于-20℃静置沉淀1小时。随后再次涡旋并以12500转/分钟、4℃条件离心15分钟，收集100 μL上清液，采用超高效液相色谱-轨道阱Exploris-120串联质谱（UPLC-Orbitrap-Exploris-120-MS/MS）联用仪进行定量分析，色谱柱选用Kinetex C18柱（2.1 mm × 100 mm，2.6 μm）。流动相由6.5 mM碳酸氢铵（A相）与乙腈（B相）组成，流速设为0.3 mL/min。梯度洗脱程序设置如下：0~1 min，2% B；1~3 min，2%~60% B；3~5 min，60% B；5~7 min，60%~100% B；7~8 min，100% B；8~8.1 min，100%~2% B；10 min，2% B。进样体积为10 μL，色谱柱温度维持在40℃，完成样品分离与分析。数据采集采用Xcalibur软件完成。质谱分析采用全扫描模式（full-scan，分辨率60000，质荷比范围60~900 m/z）与数据依赖性二级质谱（dd-MS²，分辨率15000）模式。离子源参数设置如下：喷雾电压2500 V（负离子模式），鞘气/辅助气为50/10 arb，传输管温度300℃。全扫描模式下，最大进样时间为100 ms，自动增益控制（AGC）目标值采用标准设置；数据依赖性二级质谱模式则采用自动进样时间，并设置阶梯式高能碰撞解离（HCD）碰撞能量（20%、40%、60%）。5-氟尿嘧啶与FBAL的保留时间分别为0.666 min与0.687 min。数据处理采用MS-DIAL（用于ppm计算）、Sirius（用于结果验证）以及MZmine（用于二级质谱数据导出）完成。