Agilent 7890a 5975c system

Approximately 1 g of lyophilized biomass was ground and saponified with 0.4 M KOH solution in methanol at 70 °C for 10 min. Fatty acids in the mixture were methylated using a BF₃-methanol solution at 70 °C for another 10 min after the mixture was cooled to room temperature. After methylation, 5 mL of n-hexane was added into the mixture, the top layer was then separated and placed into a phial for gas chromatography/mass spectrometry (GC/MS) analysis.
GC/MS analysis was carried out in an Agilent 7890A/5975C system (Agilent Technologies, Santa Clara, CA), which was equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The injector temperature was kept at 250 °C, with an oven temperature programmed from 50 °C to 170 °C at a rate of 10 °C min^− 1 after 5 min hold at 50 °C, from 170 °C to 250 °C at a rate of 5 °C min^− 1 after a 1 min hold time at 170 °C, and then finally maintained isothermally at 250 °C for 15 min. The carrier gas was 99.9% pure helium, with a column flow rate of 0.8 mL min^− 1. Peaks identification was performed by comparing the mass spectra to the National Institute of Standard and Technology (NIST) mass spectral library (NIST11.L). Experiments were run in triplicate, and data were shown as mean ± standard deviation (SD).

Each 1 μl of the extracted sample was analyzed by GC-MS operating in a single ion monitoring mode by Shanghai Applied Protein Technology Co., Ltd. In detail, separation and detection were made by Agilent 7890A/5975C system (Agilent) with Agilent DB-WAX column (Agilent, 0.25 μm, 0.25 mm × 30 m). The column temperature program was as follows: (1) 50 °C for 3 min; (2) temperature was gradually elevated to 220 °C in 17 min; and (3) temperature was gradually elevated to 250 °C in 2 min and maintained for 10 min. Helium was used as the carrier gas, and the flow rate was set to 1.0 mL/min. Electron impact ion source (EI) was applied for Mass spectrometry assay. The temperature was set to 280 °C for injection port, 230 °C for ion source, and 250 °C for the Inlet line. QC samples (pooled sample from an equal aliquot of each sample in the experiment) were injected with SIM mode at the beginning of the MS study and after every five injections throughout the experiment, which was used to monitor the MS performance. Finally, the mass data were analyzed by MSD ChemStation to determine the concentration of each compound.

The polysaccharide fractions DOPA-1 and DOPA-2 were methylated using methyl iodide and solid sodium hydroxide in dimethyl sulfoxide according to the method reported previously [58 (link),59 (link)]. Then, the permethylated polysaccharide samples were hydrolyzed with TFA, reduced by NaBH₄, and then O-acetylated with pyridine-acetic anhydride as partially methylated alditol acetates (PMAA), which were further analyzed by GC-MS for linkage analysis. The GC-MS analysis was performed on an Agilent 7890A-5975C system (Agilent Technology, Santa Clara, CA, USA) with a HP-5 capillary column.