Masshunter workstation software

The standard compounds of AC, HA, MA, BAC, BHC, BMA, Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, and psoralen (purity ≥98%, confirmed by LC-MS/MS, respectively) were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). Fuzi and Hongshen were purchased from Hebei Anguo Medicina Material Company (Hebei, China) and authenticated by Professor Tian-xiang Li (Tianjin University of Traditional Chinese Medicine, Tianjin, China). Acetonitrile and methanol of HPLC grade were obtained from Merck (Darmstadt, Germany). Formic acid of HPLC grade was purchased from ROE SCIENTIFIC INC (Newark, USA). Ultrapure water was purified by a Milli-Q water purification system (Millipore, Milford, MA, USA).
The LC-MS/MS system consisted of an Agilent 1200 HPLC system coupled to an Agilent 6430 triple-quadrupole mass spectrometer equipped with ESI source. All the operations and analysis of data were performed by Mass Hunter Workstation Software from Agilent Technologies (version B.04.00).

The components analysis was as follows: Aglient 1290 UHPLC instrument with auto sampler (G4226A), diode array detector (G4212A), quaternary pump (G4220A), column compartment (G1316C) and mass spectra (6550, Agilent Technologies, Palo Alto, CA, USA). Samples were separated by ACQUITY UPLCR HSS T3 (100 mm × 2.1 mm, 1.8 μm, Waters, Milford, MA, USA), the temperature was 30 °C, the detection wavelength was 254 nm, and the mobile phase was made up of acetonitrile (A) and 0.1% formic acid (B), gradient elution (v/v): 0 min, 5% A; 5 min, 20% A; 12 min 35% A; 20 min 50% A; 35 min 80% A; and 40 min 95% A. The injection volume was 2 μL. The MS parameters were shown below: analysis was carried out in negative mode and the mass range was set at 100–1200 Da. Conditions of the ESI source: Drying Gas (N₂), 10 L/min; Gas Temp, 230 °C; Nebulizer, 45 psig; Sheath Gas Flow, 12 L/min; Sheath Gas Temp, 300 °C; Capillary Voltage, 3500 V (negative mode). The sample collision energy was set at 10, 20 and 40 V. Data were processed by the MassHunter Workstation software (version B.07.00, Agilent Technologies, USA).

An 8890-gas chromatograph from Agilent Technologies (CA, USA) with a multi-purpose sampler (MPS) operating in headspace mode and a 2.5 mL syringe (Gerstel, Mülheim, Germany) were coupled to a 5977B-quadrupole mass spectrometer with an inert ion source also from Agilent. Chromatographic separation was performed using two in-line Agilent HP-5MS capillary columns (5% diphenyl-95% dimethylpolysiloxane) with 15 m × 0.25 mm I.D. × 0.25 μm, combined with a backflush system setting 0.83 min as post run time.
MassHunter Workstation software (Qualitative Analysis version B.08.00) from Agilent Technologies was used for data acquisition. StatGraphics Plus 5.1 (Statistical Graphics, Rockville, MD, USA), MS-DIAL 4.80 and SIMCA 14.1 (Umetrics, Umeå, Sweden) software were used for the processing of data. The NIST mass spectral library was used for the identification of VOCs.
For the homogenization of honey samples before analysis, an LLG-uniTEXER vortex agitator (Heathrow Scientific, Vernon Hills, Chicago, IL, USA) was used.

LC/ESI-MS was performed on a 6210 LC-TOF spectrometer coupled to an HPLC system (Agilent Technologies). All solvents used were HPLC grade (Chromasolv; Sigma-Aldrich). TFA was from Acros Organics (puriss., p.a.). Solvent A was 0.03% TFA in water; solvent B was 95% acetonitrile-5% water-0.03% TFA. Immediately before analysis, protein samples were diluted to a final concentration of 5 μM with solvent A and then desalted on a reverse-phase C8 cartridge (Zorbax 300SB-C8, 5 μm, 300 μm ID 5 mm; Agilent Technologies) at a flow rate of 50 μl/min for 3 min with 100% solvent A and subsequently eluted with 70% solvent B for MS detection. MS acquisition was carried out in the positive ion mode in the 300–3,200 m/z range. MS spectra were acquired and the data processed with MassHunter workstation software (v. B.07.00, Agilent Technologies) and with GPMAW software (v. 7.00b2, Lighthouse Data).

The HBP extract was qualitatively analyzed using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) (Agilent 1290–6545, Agilent Technologies, USA). The column used in the method was poroshell 120 column (2.1 × 100 mm, 2.7 μm, Agilent Technology, USA). The column temperature was set to 45°C. The mobile phase consisted of formic acid in water (0.1%) as solvent A and methanol as solvent B using gradient elution. The injection volume of the prepared sample was 2 μL and the flow rate was 0.3 mL/min. The solvent gradient was 0 to 1.5 min, 5% B; 5 min, 15% B; 9 min, 25% B; 16 min, 40% B; 22 min, 55% B; 28 to 30 min, 95% B; and 31 min, 5% B. Mass spectrometry was performed using electrospray source in negative ion mode with full scan, auto MS/MS mode. The capillary voltage was 3,500 V, nozzle voltage was 500 V, drying-gas temperature was 260°C, and sheath gas temperature was 360°C. Mass spectrometry data were analyzed with the MassHunter Workstation Software (Quantitative Analysis B.07.00, PCDL Manager B.07.00, and Molecular Structure Correlator, Agilent Technology, USA).

Lipids in the livers or plasma were extracted by Folch's method for LC/MS analysis20 (link). The chromatographic and mass spectrometric parameters were shown in Supplementary Table 1. LC/MS raw data were processed by MassHunter Workstation software (version B.04.00 Qualitative Analysis, Agilent Technologies) and Mass Profiler Professional software package (version 2.2, Agilent Technologies) as described20 (link). Authentic commercially available standards (Sigma-Aldrich), as shown in supplementary Table 2, were used to confirm the identities of the lipid species. Hexadecanoic-15,15,16,16,16-d5 acid was used as internal standard in the targeted lipidomics20 (link).

Polyphenolic compounds were identified by using an Agilent 6520 Accurate-Mass quadrupole time of flight (QTOF) machine described by Suleria et al. [18 (link)] after modifications. Column, gradient, chromatographic, and other machine conditions were the same as we reported [11 (link),19 (link)]. MassHunter Workstation Software (version B.06.00) (Agilent, Santa Clara, CA, USA) was used to extract and identify phytochemicals. The mass spectra of twenty-four external standards were also obtained and standard equations were generated as described by Ali et al. [20 (link)].