2.8. Organic acid quantification by LC-MS/MS

LC–MS/MS-based organic acid analysis was performed as described previously with some modifications (Kim et al., 2020; Song et al., 2020). The culture supernatant sample was filtered using a polyvinylidene fluoride membrane syringe-driven 0.45-μm pore size filter (SLHVX13NL, Millex-DV, Millipore, MA, USA) to remove cell debris. Next, the filtered sample was transferred to a microtube and diluted 10-fold with water. Then, 50 μl 50% acetonitrile, 40 μl 100 mM N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide (EDC, 1769, Sigma-Aldrich, MO, USA), 40 μL 100 mM Girard’s reagent T (G900, Sigma-Aldrich, MO, USA), and 10 μL 1 mM sodium [²H₇]butyrate (D5372, CDN ISOTOPES, QC, Canada) were added in 20 μl diluted sample and mixed. The mixture was incubated at 40°C for 1 h, and the samples were diluted 20-fold with 50% acetonitrile. The prepared samples were stored at −20°C until further analysis.

For LC–MS/MS analysis, Acquity UPLC H-Class (Waters, MA, USA) combined with an LTQ XL™ linear ion trap mass spectrometer (Thermo Fisher Scientific, MA, USA) was used. The sample (5 μL) was injected into a Zorbax HILIC plus column (4.6 mm, 100 mm, and 3.5 mm; Agilent, CA, USA). Solvent A comprised water, 20 mM ammonium acetate, and 20 mM acetic acid, while solvent B comprised 100% acetonitrile. The LC gradient was 0 min, 70% B; 1 min, 70% B; 10 min, 30% B; 15 min, 30% B; 15.1 min, 70% B; and 20 min, 70% B. The flow rate was 0.3 mL/min. The NCE was 15–30 eV. For the analysis of organic acids, the previously reported MS/MS detection parameters of 5 GT-labeled short-chain fatty acid and lactate detection parameters (m/z of precursor ion: 204.1; m/z of product ion: 100.1; NCE: 30) were used.