FFAs were extracted from mouse feces using a modified version of the Bligh and Dyer’s method (LipidALL Technologies Co., Ltd, Changzhou, China). Briefly, fecal samples were homogenized with 750 µL of chloroform: methanol 1:2 (v/v) and 10% deionized water, and incubated at 4 ℃ for 30 min. The samples were centrifuged after addition of 250 µL of chloroform and 350 µL of deionized water. Lipid in the lower organic phase after centrifugation was extracted twice. After that, the total extract was collected and dried in the SpeedVac under OH mode.
Agilent 1290 UPLC combined with a triple quadrupole/ion trap mass spectrometer (6500 Plus Qtrap; SCIEX) was used for FFAs analysis. Normal phase (NP)-HPLC with a Phenomenex Luna 3 µm-silica column (internal diameter 150×2.0 mm) was used for lipids separation. The conditions as follows; chloroform: methanol: ammonium hydroxide (89.5:10:0.5) was used as mobile phase A, and chloroform: methanol: ammonium hydroxide: water (55:39:0.5:5.5) was used as mobile phase B. D31-16:0 (Sigma-Aldrich) and d8-20:4 (Cayman Chemicals) was used as internal standards for FFAs quantitation.
Agilent 1290 UPLC combined with a triple quadrupole/ion trap mass spectrometer (6500 Plus Qtrap; SCIEX) was used for FFAs analysis. Normal phase (NP)-HPLC with a Phenomenex Luna 3 µm-silica column (internal diameter 150×2.0 mm) was used for lipids separation. The conditions as follows; chloroform: methanol: ammonium hydroxide (89.5:10:0.5) was used as mobile phase A, and chloroform: methanol: ammonium hydroxide: water (55:39:0.5:5.5) was used as mobile phase B. D31-16:0 (Sigma-Aldrich) and d8-20:4 (Cayman Chemicals) was used as internal standards for FFAs quantitation.