Autosampler vial

The frozen AF samples stored at -80 °C were thawed on ice. Then, they were mixed well and centrifuged at 1,000 g for 3 min at 4 °C. Each 100 μL of AF sample was aliquoted into a pre-cooled Eppendorf tube and 10 μL of the internal standard solution was added. For metabolite extraction, each 200 μL of pre-cooled methanol: chloroform (3:1, v/v) was used. After centrifugation (13,000 g, 20 min, 4 °C), the supernatant was transferred into an auto-sampler vial (Agilent Technologies, Foster City, CA, USA). Quality control (QC) samples were prepared by mixing the remaining supernatant from each AF sample. To remove the chloroform solvent, all the samples were centrifuged for 5 min in a vacuum centrifuge concentrator (Labconco, Kansas City, MO, USA), and then transferred to a freeze dryer (Labconco, Kansas City, MO, USA) and completely lyophilized. Dichloromethane was added to ensure complete dryness of the samples, followed by high-purity nitrogen (Parker Balston, Lancaster, NY, USA) filling in the dried powder at room temperature. Untargeted metabolite analysis was performed on the XploreMET platform (Metabo-Profile, Shanghai, China). Briefly, 50 μL of methoxyamine (20 mg/mL in pyridine) was added to each dried sample at 30 °C for 2 h and then mixing with 50 μL of MSTFA (1% TMCS) containing FAMEs at 37.5 °C for 1 h.

The untargeted metabolomics profiling was implemented on XploreMET platform (Metabo-Profile, Shanghai, China). The sample preparation was conducted as their published methods with minor modifications [24 , 25 (link)]. In brief, the plasma samples were centrifuged at 3000×g and 4 °C for 5 min (Microfuge 20R, Beckman Coulter, Inc., Indianapolis, IN, USA) after thawing to separate debris or a lipid layer. Metabolites were extracted from plasma samples (50 μL) with 10 μL of internal standard (0.5 mM 4-Chlorophenylalanine) and 175 μL of pre-chilled methanol: chloroform (3:1) followed by centrifugation at 14, 000×g for 20 min at 4 °C. Then each 200 μL of the supernatant was transferred into an autosampler vial (Agilent Technologies, Foster City, CA, USA). The resting supernatant from each sample was pooled to prepare quality control samples. Following solvent evaporation and lyophilization, the dried samples were derivatized with 50 μL of methoxyamine (20 mg/ml in pyridine) for 2 h, followed by silylanization with 50 μL of MSTFA (1% TMCS) for 1 h prior to injection. Above two steps were performed by a robotic multipurpose sample MPS2 with dual heads (Gerstel, Muehlheim, Germany).

Peripheral blood was obtained from mice at the end of study and was incubated for 30 min at room temperature to allow for clotting then centrifuged at 2,500 × g for 10 min to extract the serum from clot. Serum aliquots were stored at −80°C until the time of analysis. Feces samples were collected from ileocecal junction after sacrifice and stored at −80°C until DNA extraction from feces.
The serum sample preparation procedures were described previously (Wang et al., 2013 (link)). In brief, the serum samples were centrifuged at 4°C, 3,000 × g to separate debris or lipid layer. Each 50 μl aliquot was mixed with 10 μl internal standard, and 175 μl pre-chilled methanol was added. Then the mixtures were kept at −20°C for 20 min and centrifuged for 20 min at 4°C 14,000 × g. The supernatant was transferred into an autosampler vial (Agilent Technologies, Foster City, CA, United States) separately and was evaporated briefly, following by lyophilizing with a FreeZone freeze dryer (Labconco, Kansas City, MO, United States). The dried samples were then derivatized with 50 μl methoxyamine at 30°C for 2 h, followed by adding 50 μl N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) containing fatty acid methyl esters (FAMEs) as retention indices and further incubating at 37.5°C for 1 h. Finally, the derivatized samples were injected into the GC-TOF/MS for analysis.

All samples were weighed on an electronic balance (AL204, Mettler Toledo) and were homogenized in two volumes of cold purified water using an electric homogenizer (IKA T10, Germany) in an ice bath. Then, 500 µL of homogenate was placed in an Eppendorf tube and 1500 µL of 50% methanol and 50% acetonitrile (v/v = 1:  1) solution containing 0.1% formic acid was added; the solution was then vortexed for 3 min. The precipitate and the supernatant were then separated by centrifugation at 20 000 (×g) for 20 min at 4°C. The supernatant (1500 µL) was evaporated to dry under a stream of nitrogen gas at 37°C and was then re-suspended in 200 µL of methanol. The mixture was vortexed well and centrifuged at 20 000 (×g) for 10 min at 4°C. Then, 150 µL of the supernatant was transferred to an autosampler vial (2 ml; Agilent) with a vial insert (250 µL insert, polypropylene, Agilent) and a 5 µL aliquot was injected into the LC/MS-MS system for analysis.

All chemicals were purchased from commercial suppliers and used as received. 2,2,2-Trichloroethoxycarbonyl chloride (Troc-Cl), ammonium hydroxide and dichloromethane were purchased from Sigma-Aldrich (St. Louis, MO.). Potassium bicarbonate was purchased from Acros Organics (Westchester, PA.). Autosampler vials were purchased from Agilent Technologies (Santa Clara, CA.). Fentanyl and acetylfentanyl were synthesized using published protocols [11 (link)]. Thin layer chromatography (TLC) was used to monitor the production of amine-containing intermediates leading to the synthesis of fentanyl and acetylfentanyl using Merck 60-F254 sheets and detection accomplished with UV light (λ = 254 nm) in conjunction with CAM [36 (link), 37 (link)] and iodine vapor [38 (link), 39 (link)]. All fentanyls as well as the Troc-norfentanyl and Troc-noracetylfentanyl standards were purified by flash column chromatography using a Biotage Isolera purification system.