Agilent 6460 triple quadruple mass spectrometer

The mass spectrometric detection was performed on an Agilent 6460 triple-quadruple mass spectrometer (Agilent Technologies, Singapore) with an electrospray ionization source. It was operated in the negative ion detection mode because of its higher sensitivity than that in the positive ionization mode on multiple reaction monitoring (MRM). The data were acquired and processed using Mass Hunter Workstation B.06.00 software (Agilent Technologies, Singapore). The mass spectrometric parameters of each compound are summarized in Table 1 [12 (link)]. The MS spectra of the six anthraquinones are shown in Figure 2. The source parameters were also optimized as follows: a drying gas flow and temperature at 6 L/min and 350 °C were used, respectively; the sheath gas flow and temperature were maintained at 12 L/min and 350 °C, respectively; the nebulizing gas (N₂) pressure was set at 25 psi; and the capillary and nozzle voltages were set at 3500 V and 500 V, respectively.

An Agilent 1200 HPLC system (Agilent, USA) with electrospray ionization coupled to an Agilent 6460 triple quadruple mass spectrometer (LC-ESI-MS/MS) was used to confirm the structures of the GSLs. The HPLC conditions were the same as those mentioned in the previous section, and the mass conditions are listed in Table 6. Compounds with m/z = 75 and featuring [M-G-H]⁻ molecular ion peaks were selected as candidate compounds [90 (link), 91 ]. The positive ion peak, such as [M-G + H]⁺, was used for identification [51 (link), 92 (link)]. For MS/MS conditions, the fragmentor voltage was optimized by approximately 1/3 of the molecular weight, and the collision energy number was set to approximately 1/15 of the given molecular weight.Table 6

LC-MS/MS conditions

Items	Parameters
Gas Temp	300 °C
Gas Flow	10 L/min
Nebulizer	45 psi
Sheath Gas Temp	350 °C
Sheath Gas Flow	11 L/min
Capillary	3000 V(+)/3000 V(−)
Nozzle Voltage	500 V(+)/500 V(−)

To measure the levels of SCFAs in the fecal samples, we followed the methods by Han et al. [31 (link)]. First, to measure the dry weight of the fecal samples, 2 g was homogenized with 5 mL of acetonitrile and dehydrated using a high-speed vacuum centrifuge. The samples were then stored at −80 °C at a concentration of 2 mg/mL until further analysis. After being centrifuged at 4000× g, 20 °C for 10 min, the clear supernatants were collected for measuring the concentrations of SCFA. The supernatant was then combined with 3-nitrophenylhydrazine (3-NPH), N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide (EDC), and an internal standard solution and heated to 40 °C for 30 min to derivatize the clear supernatant. The derivatized sample was pretreated for fecal SCFA analysis by adding formic acid to stop the reaction. The SCFAs were then determined using an Agilent 6460 Triple Quadruple Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) and a 1260 Infinity UPLC system (Agilent Technologies, USA). The samples were separated using a Poroshell 120 SB-C18 column (Agilent Technologies, USA) with the mobile phase A being water containing 0.1% formic acid and mobile phase B being acetonitrile containing 0.1% formic acid, with a total flow rate of 200 L/min. The temperature of the thermostat column compartment was maintained at 60 °C throughout the analysis.