Uflc xr device

HPLC analyses were carried out using a Shimadzu UFLC XR device at 40°C, a flow rate of 1.0 mL min⁻¹, injection volume was 10 μL, detection wavelength 254 nm. The stationary phase was a Phenomenex® HyperClone ODS (C18) column, 120 Å, 5 μm, 150 × 4.60 mm; mobile phase A: H₂O + 0.9% AA + 0.1% FA; mobile phase B: CH₃CN + 0.9% AA + 0.1% FA; 0 min: 90% A to 30 min: 2% A, 30-35 min: 2% A.

The LC–MS analyses were performed on a UFLC-XR device (Shimadzu, Japan) coupled to a QTRAP 5500 MS/MS hybrid system triple quadrupole/linear ion trap mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with a Turbo VTM ion source. Instrument control and data acquisition were performed using the Analyst 1.5.2 software. The RP-HPLC separation was carried out on the same column used for the RP-HPLC analyses (§ 2.4.1). The elution was performed with the same gradient, previously described, using formic acid (0.1%) instead of trifluoroacetic acid (0.1%) for the RP-HPLC analyses. MS analysis was carried out in positive ionization mode using an ion spray voltage of 5500 V. The nebulizer gas (air) and the curtain gas (nitrogen) flows were set at 30 psi. The Turbo VTM ion source was set at 550 °C with the auxiliary gas flow (air) set at 50 psi.
The MS/MS analyses were completed with the BioAnalyst 1.5.1 and Peaks 7 software.

Plasma concentrations of the bile acids (BAs) were determined after protein precipitation with iced methanol as previously published [34 (link)]. BAs from the cecal content were quantified after extraction on samples lyophilized at −80 °C to avoid bacterial BA. In both biological samples, the 27 BA species were quantified by high-performance liquid chromatography (UFLC-XR device, Shimadzu, Kyoto, Japan) coupled to tandem mass spectrometry (MS/MS) (QTRAP 5500 hybrid system, equipped with a Turbo VTM ion source, Sciex, Foster City, CA, USA) using five deuterated BAs (D4-CA, D4-GCA, D4-TCA, D4-CDCA, D4-GCDCA) as internal standards. Plasma and cecal content BA concentrations were expressed in nmol/l and as a percentage of total BAs, respectively. Ratio and total values were determined according to formulas presented in Table S4.

When necessary, purification from one fraction was performed by HPLC using a Shimadzu UFLC XR device equipped with a PDA detector (SPD-M20A, Shimadzu, Kyoto, Japan). A Spherisorb ODS2 column (5 μm, 250 × 4.6 mm, Waters, Milford, MA) was used for solute separation, and the mobile phase consisted of a mixture of acetonitrile 100% (A) and ultrapure water (B). The following linear gradient was applied: t = 0 min 100% B; t = 10 min 100% A; t = 12 min 100% A; t = 15 min 100% B. Compounds were detected at 254 nm and collected. When needed, some of them were submitted to acid hydrolysis treatment (1 N HCl, 110 °C for 1 h) before structural elucidation.

When required, the purification of solutes from a specific fraction was carried out by HPLC using the Shimadzu UFLC XR device equipped with a PDA detector (SPD-M20A, Shimadzu, Kyoto, Japan). For solute separation, a Spherisorb ODS2 column (5 µm, 250 × 4.6 mm, Waters, Milford, MA, USA) was used, and the mobile phase consisted of a mixture of 100% acetonitrile (A) and ultrapure water (B). The purification process involved the following linear gradient: t = 0 min 100% B; t = 10 min 100% A. The compounds were detected at 254 nm and collected for acid hydrolysis treatment (1 N HCl, 110 °C for 1 h) prior to structural elucidation.