Ultra high performance liquid chromatography

The metabolites in the cell lysates and tissues from mice were measured by LC-MS/MS (ACQUITY UPLC) in the laboratory of Prof. Yi Zhu at Tianjin Medical University as they previously described [26 (link)]. Briefly, 100 mg of tumour tissues, livers, or lungs were homogenized before lipid extraction. HUVECs were lysed by repeated freeze–thawing cycles and then pre-processed in methanol. After centrifugation, the supernatant was extracted by ethyl acetate twice, and then the upper organic phase was evaporated. The residue was then dissolved in 100 μl 30% acetonitrile. The resulting sample was then subjected to ultra-high-performance liquid chromatography (Waters, Milford, MA) with a 5500 QTRAP hybrid triple-quadruple linear ion trap mass spectrometer (AB Sciex, Foster City, USA) equipped with a Turbo Ion Spray electrospray ionization source.

Then, 100 μL serum or 200 mg cecal contents were extracted with 300 μL methanol containing an internal standard mixture, evenly mixed for more than 1 min, and stayed on ice for 30 min. Samples were centrifuged with 12,000× g for 30 min at 4 °C. The supernatant was transferred for bile-acid analysis. The analysis was performed using Waters ultra-high-performance liquid chromatography coupled with a Waters Xevo TQS MS (Waters Corp., Milford, MA, USA). For bile-acid analysis, chromatographic separation was performed on a Waters ACQUITY BEH C8 column (2.1 mm × 100 mm × 1.7 μm). The column temperature was maintained at 60 °C. For optimized parameters, mobile phase A was 10% acetonitrile with 0.01% formic acid, and mobile phase B was isopropanol/acetonitrile (50:50, v/v) with 0.01% formic acid. Mass analysis was performed using the Waters Xevo TQ-S system in positive-ion ESI mode. The capillary voltage was set at 1.5 KV. The desolvation gas flow rate was set at 1000 L/h, and the cone gas flow was maintained at 150 L/h. The desolvation and source temperatures were set at 600 °C and 150 °C, respectively. The QC sample (laboratory quality control) and mix QC sample (a mixture of all samples) were prepared for analysis during the analytical runs after every 10th sample.

Wild OS from Baima Snow Mountains were collected in June 2021. The bacterial membrane was first washed with distilled water, and OS was cut from the base of the stroma into two parts, the stroma and sclerotia. The stroma was labeled as OSBSz-x and the sclerotia was labeled as OSBSh-x. Three biological replicates were used for each group.
Instruments for widely targeted metabolomics include ultrahigh-performance liquid chromatograph (Sciex, Framingham, MA, USA), a high-sensitivity mass spectrometer (Sciex, Framingham, MA, USA), and a centrifuge (Thermo Scientific, Waltham, MA, USA), and a water purification system (Merck Millipore, Burlington, MA, USA). Instruments for untargeted metabolomics include ultrahigh-performance liquid chromatography (Waters, Milford, MA, USA), a high-resolution mass spectrometer (Thermo Scientific, Waltham, MA, USA), a low-temperature high-speed centrifuge (Eppendorf, Hamburg, Germany), a vortex (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen, China), a water purification system (Millipore, Burlington, MA, USA), and a refrigerated vacuum concentrator (Gene company limited, Hong Kong, China).

Free chlorine, ClO₃⁻, and ClO₄⁻ were quantified in selected samples. The Concentration of HClO was measured by spectrophotometer at 510 nm (Beckman Coulter DU 800, Brea, CA). A 2.5-mL aliquot sample was immediately mixed with 0.25-mL DPD solution (8.0 mM). DPD is oxidized by HOCl to show a red color. ClO₃⁻ and ClO₄⁻ were analyzed using a Waters (Milford, MA) ultra-high performance liquid chromatography with an electrospray ionization (ESI) source (UPLC-MS/MS). Detailed UPLC-MS/MS analytical parameters can be found in Text S2. Quantification of the ClO₃⁻ and ClO₄⁻ was based on multipoint standard calibration.

An ultrahigh-performance liquid chromatography (Waters, United States) was used for separation and detection. A Waters ACQUITY UPLC HSS T₃ column (2.1 × 100 mm, 1.8 μm) was employed at 35℃ with a flow rate of 0.3 ml min⁻¹. The mobile phase consisted of acetonitrile (A) and water containing 0.1% FA (B). The injection volume of each sample was 3 μl. The following linear gradient was used: 0–4.5 min: 30–90% A and 4.5–5.5 min: 95% A. MS conditions were the same as those in “2.3.2.” The MRM transitions, cone voltage, and collision voltage of the target compounds and IS are shown in Supplementary Table S1.

Erythrocyte membrane lipidomics analysis was performed by ultra-high-performance liquid chromatography (Waters, Milford, MA) coupled with a triple quadrupole time-of-flight mass spectrometer UPLC-Q-TOF/MS (AB SCIEX 5600 Q-TOF, Framingham, MA) system [25 (link)]. For separation, a C8 ACQUITY column (2.1 mm × 100 mm × 1.7 µm, Waters, Milford, USA) was used. Mobile phases were as follows: A was 60% acetonitrile (acetonitrile/H₂O = 6 : 4, 10 mM ammonium acetate) and B was isopropanol/acetonitrile = 9 : 1, 10 mM ammonium acetate; flow rate 0.26 ml/min; 32% B for 1.5 min, linear increase to 85% B in 14 min, then increase to 97% B, kept for 2.4 min, and then decrease 97% B to 32% B within 0.1 min and maintained 1.9 min. Mass spectrometry was recorded under both positive and negative electron-spray ionization (ESI) modes, voltages 4500 V for ESI− and 5500 V for ESI+, respectively. The declustering potential was 100 V, curtain gas was 35 psi, and interface heater temperature was 600°C (ESI−) and 500°C (ESI+), respectively. For MS/MS analysis, information-dependent acquisition (IDA) was applied with the collision energy of 10 V.

The β-carotene in the flesh was extracted as described by Ma et al. (2018 (link)). The carotenoids were analyzed using the Waters Ultra-high-performance liquid chromatography (Milford, MA, USA) with a photodiode array detector and a single quadrupole mass spectrometer detector in series (6120 Quadrupole, Agilent, Santa Clara, CA, USA). The column used was a YMC carotenoid (C30) column (Wilmington, NC, USA). The eluent phases were as follows: mobile phase A, acetonitrile:methanol = 3:1 (v/v); and phase B, 100% methyl tert-butyl ether (MTBE). Each eluent contained 0.01% butylated hydroxytoluene (BHT). Gradient elution was performed as follows: 0–2 min, 85:15 A:B; 2–4 min, 75:25 A:B; 4–7 min, 40:60 A:B; 7–10 min, 40:60 A:B; 10–13 min, 5:95 A:B; 13–23 min, 85:15 A:B. The flow rate was 0.8 ml/min, and the injection volume was 5 μl.