Q exactive orbitrap mass analyzer

For ultra-high-performance LC hyphenated to Quadrupole-Orbitrap high-resolution MS (UHPLC-Q-Orbitrap-HRMS) analysis, the methodology used was as previously described [18 (link)]. The chromatographic separation was performed using an Ultimate 3000 XRS UHPLC system (Thermo Fisher Scientific, San José, CA, USA). An Acquity UPLC HSS-T3 column (1.8 μm, 150 mm × 2.1 mm) (Waters, Manchester, UK), kept at 45 °C, was used, to which a binary solvent system consisting of ultrapure water (A) and acetonitrile (B), with both acidified with 0.1% formic acid, was applied at a constant flow rate of 0.4 mL/min. All solvents used were of LC-MS grade and obtained from Fisher Scientific and VWR International (Merck). Ultrapure water was obtained by usage of a purified-water system (VWR International, Merck). Mass analysis was performed on a Q-ExactiveTM Orbitrap mass analyzer (Thermo Fisher Scientific) that was equipped with a heated electrospray ionization (HESI II) source, operating in polarity switching mode. Hereby, full-scan events were applied at a mass resolution of 70,000 full width at half maximum. A pool of all extracts (n = 150) was used to make quality control (QC) samples for instrument conditioning and data normalization. Experimental samples were run in a randomized order, except for QC samples, which were analyzed in duplicate after every nine experimental samples.

The UHPLC-Q-Orbitrap-MS method as used in this study was adopted from Vanden Bussche, et al. [26 (link)], as optimized by De Paepe et al. [27 (link)]. The chromatographic separation was performed with an Ultimate 3000 XRS UHPLC system (Thermo Fisher Scientific). Analysis was performed on a Q-Exactive^TM Orbitrap mass analyzer (Thermo Fisher Scientific) that was equipped with a heated electrospray ionization (HESI II) source operating in polarity switching mode. An Acquity UPLC HSS T3 column (1.8 μm, 150 × 2.1 mm) (Waters), kept at 45 °C, was used, to which a binary solvent system consisting of ultrapure water (A) and acetonitrile (B), both acidified with 0.1% formic acid, was applied at a constant flow rate of 0.4 mL/min. A gradient profile with following proportions (v/v) of solvent A was applied: 0–1.5 min at 98%, 1.5–7.0 min from 98% to 75%, 7.0–8.0 min from 75% to 40%, 8.0–12.0 min from 40% to 5%, 12.0–4.0 min at 5%, 14.0–14.1 min from 5% to 98%, followed by 4.0 min of re-equilibration. A pool of all extracts (n = 25) was used as quality control (QC) samples for instrument conditioning and data normalization. All solvents used were of LC-MS grade. Experimental samples were run in a randomized order (except for QC samples, which were analyzed in duplicate after every ten experimental samples).

The UHPLC-Orbitrap-MS method that was used in this study was adopted from^{54 (link)} as previously optimized^{55 (link)}. An external standard mixture containing ca. 300 metabolites (including amino acids, monocarboxylic acids, phenols, multi-carboxylic acids, amines, carbohydrates, polyols, short chain fatty acids, inorganic acids, bile salts, and N-compounds) was used to assess instrumental performance and execute targeted profiling. A pool of all extracts (n = 84) was used to make quality control (QC) samples for instrument conditioning (external QC samples) and data normalization (internal QC samples). The Q-Exactive^TM Orbitrap mass analyzer (Thermo Fisher Scientific, San Jose, CA) was equipped with a heated electrospray ionization (HESI II) operating in polarity switching mode. An Acquity UPLC HSS T3 column (1.8 μm, 150 mm × 2.1 mm) (Waters, Zellik, Belgium) was used, whereby a binary solvent system using ultrapure water (A) and acetonitrile (B), both acidified with 0.1% formic acid, was applied at a constant flow rate of 0.4 mL min⁻¹. All Solvents used were of LC-MS grade. Experimental samples were run in a randomized order (except for quality control samples, which were analyzed in duplicate after every nine experimental samples).