2.7. Gas Chromatography–Mass Spectrometry (GC-MS)

The monosaccharides Glc, Gal, Mann, Xyl, apiose, Myo-inositol (Internal Standard, IS), and the pyridine, hexane and methanol/3 M HCl solution were purchased from Sigma Aldrich. Tri-Sil HTP Reagent was purchased from Thermo Scientifics.4.9.2. Derivatization of polysaccharides. TMS (trimethylsilyl) derivatization was performed on previous reports with appropriate modifications [26]. Polysaccharides samples (2 mg) and monosaccharides standards were treated using the same procedure. An aliquot (100 µL) of the standard stock solution of 1 mg mL⁻¹ of each monosaccharide was dried under nitrogen gas flow. A mixture of 2 mL MeOH/3M HCl was added to the samples of PcSPs, and a mixture containing the standard monosaccharides. The resultant solution was kept at 80 °C for 24 h [33], subsequently washed with methanol and dried under nitrogen gas flow. The trimethylsilyl reaction was then carried out with 200 µL of Tri-Sil HTP. The sample vials were heated at 80 °C for 1 h. The derivatized samples were cooled to room temperature and dried under a stream of nitrogen. The dry residue was extracted with hexane (2 mL) and centrifuged. Finally, the solution containing silylated monosaccharides was concentrated and reconstituted in hexane (200 µL), filtered and transferred to a GC-MS autosampler vial. Sample preparation and analyses were performed in triplicate.

GC/MS analyses were carried out using a gas chromatography Trace GC (Thermo Scientifics), autosampler Tri Plus and DSQ mass spectrometer quadrupole (Thermo Scientifics). The column was ZB-5 Zebron, Phenomenex (5% Phenyl, 95% Dimethylpolysiloxane) with dimensions of 30 m × 0.25 mm i.d. × 0.25 µm. The column temperature program started at 80 °C (held 2 min) and underwent a gradient of 5 °C/min to reach a final temperature of 230 °C. The carrier gas was helium (flow 1.2 mL/min). The injection volume was 1 µL in splitless mode at 250 °C. The source and MS transfer line temperature were 230 °C. The mass spectrometer was set for a Select Ion Monitoring (SIM) program in electron ionization mode (EI) at 70 eV. The TMS-derivatives were identified by characteristic retention times and mass spectrum compared to those of the standards that were used for the identification of monosaccharides. The compounds were identified by comparing the mass spectra with those in the National Institute of Standards and Technology (NIST 2014) library.