Triple quadrupole 6 470 mass spectrometer

MS analysis of bile acids was performed on an Agilent 1,260 series HPLC system (Agilent Technologies, Santa Clara, CA, United States) equipped with a quaternary pump, degasser, auto sampler and thermostatically controlled column compartment. Chromatographic separation was performed on an Agilent Poroshell 120 EC-C18 (3.0 mm × 150 mm, 2.7 micron) at 37°C. The structures of bile acids are shown in Figure 1 and Table 1. The mobile phase consisted of 5 mmol/L ammonium acetate containing 0.1% formic acid (solvent A) and methanol (solvent B). A gradient elution program was used as follows: linear gradient from 0 to 2 min, 25% B–46% B; 2–27 min, 46% B–70% B; 27–35 min, 70% B−95% B; 35–38 min, 95% B–98% B. The flow rate was 0.3 mL/min and the injected sample volume was 3 µL. Detection was achieved using an Agilent triple quadrupole 6,470 mass spectrometer (MS) with an electrospray ionization source. The main working parameters for MS were set as follows: drying gas (N₂) flow rate, 10 L/min; drying gas temperature, 300°C; nebulizing gas (N₂) pressure, 45 psi; capillary voltage, 3,000 V; quadrupole temperature, 300°C. Fragment ion spectra were recorded by negative electrospray ionization in the multiple reaction monitoring mode (MRM) and MS parameters, as shown in Table 2.

The levels of SCFA (acetic acid, propionic acid, and butyric acid) were measured using a 3-nitrophenylhydrazine derivatization strategy. About 100 mg of feces were extracted with 20 × 70% methanol (m/v) and then processed with TissueLyzer after adding zinc beads. The fecal samples were then centrifuged at 14,000 rpm at 4°C for 15 min. About 200 μL of upper supernatant was transferred to new tubes for LC-MS analysis. An Agilent 1290 Infinity II UPLC system coupled with a triple quadrupole 6470 mass spectrometer was used for targeted metabolomic profiling. A Waters BEH C₁₈ column (50 mm × 2.1 mm, 1.7 μm) with a precolumn was used. The MS data were collected and processed using Agilent software. The chromatographic condition was used as described previously by Dei Cas et al 22 , a and mass spectrometry detection was performed as described in our previous study 23 (link).

Equal numbers of HEK293T cells expressing soluble GFP or overexpressing GFP fusions to SPNS1 or mutant SPNS1 were incubated in 100 μl of transport assay buffer with the indicated concentration of d7-LPC (18:1) (Avanti) for 20 min at 37°C. The composition of transport assay buffers was as follows: pH 5: 25 mM sodium acetate, 5 mM glucose, 1 mM MgCl, and 150 mM NaCl; pH 7: 25 mM Hepes, 5 mM glucose, 1 mM MgCl, and 150 mM NaCl. Following incubation, cells were placed on ice and immediately washed twice with 0.5% fatty acid–free BSA in PBS and once with PBS. Lipids were extracted in acetonitrile:isopropanol:water of 13:6:1 (v/v/v) for 15 min and spun at 20,000g for 10 min at 4°C to remove cell debris. Lipid extracts were analyzed on the Agilent Triple-Quadrupole 6470 Mass Spectrometer as described below.