Accucore rp ms column

The dried extract was re-dissolved under the acid hydrolysis method as previously reported [51 (link)]. In this condition, the glycosidic bond between sugar and phenolics is disrupted, leading to its aglycone. Briefly, the extract was re-dissolved in formic acid and 62.5% (v/v) methanol containing tert-butyl hydroquinone, then shaken at 80 °C for 2 h and filtered through a 0.22 µm polytetrafluoroethylene (PTFE) filter. The extract was then loaded to an Accucore RP-MS column (a 2.1 mm × 100 mm, 2.6 μm column (Thermo Fisher Scientific, Bremen, Germany), which was connected to the LC–ESI–MS/MS system consisting of a Dionex Ultimate 3000 series ultra-high-performance liquid chromatograph (UHPLC) system, a diode array detector, a TSQ Quantis Triple Quadrupole mass spectrometer (MS), and a Chromeleon 7 chromatography data system (version 7.2.9.11323, Thermo Fisher Scientific, Bremen, Germany).
Acetonitrile (A) and 0.1% v/v formic acid in Milli-Q water (B) were used as mobile phase. A gradient solvent system was set as follows: 0.0–0.8 min, 10–80% A; 8.0–8.1 min, 80–10% A; 8.1–10.0 min, 10% A, at a flow rate of 0.5 mL/min. Supplementary Tables S1 and S2 show a list of authentic standards with parameters and validations, respectively.

Chromatographic analysis was performed using a UPLC Vanquish Flex Binary system (Thermo Fisher Scientific, Waltham, MA, USA). The column used for chromatographic separation was an Accucore RP-MS column (2.1 mm × 150 mm, 2.6 μm, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase was constituted of aqueous formic acid 0.1% as phase A and acetonitrile formic acid 0.1% as phase B using gradient elution at a flow rate of 0.4 mL/min. The following gradient was applied: 0–2 min, 1% B; 2–20 min, 1–75% B; 20–20.1 min, 75–100% B; 20.1–22 min, 100% B; 22–22.1 min, 100–1% B and 22–26 min, 1% B. The injection volume was 10 μL and the column temperature was maintained at 30 °C. All samples were injected in triplicate in a randomized order to avoid deviations caused by injection order. The QC sample was injected at the beginning and the end as well as along the analytical run.

Briefly, chromatographic separation was performed using a Shimadzu Nexera 2 LC system (Shimadzu Corporation, Marne-la-Vallée, France) equipped with a thermostated column compartment and a thermostated microwell plate autosampler with a six-port micro-switching valve. LC separation was performed on an Accucore™ RP-MS column (100 × 2.1 mm, 2.6 μm solid core, Thermo Scientific) at a flow rate of 0.2 mL/min at 60 °C. The mobile phase was a gradient of 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B) programmed as follows: 0-0.5 min, 30% B; 0.5-8 min, 30 to 50% B; 8-9 min, 50 to 95% B; 9-11 min, 95% B; 11-11.5 min, 95 to 30% B; 11.5-15 min, 30% B. Detection was carried out with a LCMS8060 mass spectrometer (Shimadzu) in the positive ionization mode. Of the four optimized MRM transitions per analyte, the most intense and specific Q1/Q3 pair was selected for quantification: 194 > 137 for acetic acid, 197 > 137 for D4-acetate, 208 > 137 for propionic acid, 210 > 137 for D2-propionate, 222 > 137 for butyric acid and 229 > 137 for D7-butyrate. The MRM parameters for all analytes and their internal standards are listed in Table S1. The concentration of each SCFA was determined by calculating its corresponding peak area ratio to that of the IS using a linear regression with 1/x weighting to the calibration curve.