Apiose

EgP samples (2 mg) and monosaccharide standards were treated with the same procedure. First, 100 µL of the standard stock solution of 1 mg mL⁻¹ of each monosaccharide was dried under nitrogen gas flow. Second, the samples of polysaccharides, and a mixture containing the standard monosaccharides included in the Internal Standard (IS), were methanolyzed in 2 mL metanol/3 M HCl at 80 °C for 24 h. The monosaccharides, glucose, galactose, rhamnose, fructose, mannose, xylose, apiose, and myo-inositol (IS) (Sigma-Aldrich, St. Louis, MO, USA), as well as pyridine, hexane and metanol/3 M HCl solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Then, the saccharides were washed with methanol and dried under nitrogen gas flow. Third, the Trimethylsilyl reaction was accomplished with 200 µL of Tri-Sil HTP (Thermo Fisher Scientific, Franklin, MA, USA). Each vial with the sample was heated at 80 °C for 1 h. The derived sample was cooled to room temperature and dried under a stream of nitrogen. Fourth, the dry residue was extracted with hexane (2 mL) (Sigma-Aldrich, St. Louis, MO, USA), and centrifuged. Finally, the hexane solution containing silylated monosaccharides was concentrated and reconstituted in hexane (200 µL), filtered and transferred to a GC-MS autosampler vial (Thermo Fisher Scientific, Franklin, MA, USA). Sample preparation and analyses were performed in triplicate.

Polysaccharides samples (2 mg) and monosaccharides standards were treated with the same procedure. First, 100 µL of the standard stock solution of 1 mg mL⁻¹ of each monosaccharide was dried under nitrogen gas flow. Second, the samples of polysaccharides, and a mixture containing the standard monosaccharides included in the IS (Internal Standard), were methanolized in 2-mL methanol/3-M HCl at 80 °C during 24 h. The monosaccharides glucose, galactose, rhamnose, fructose, mannose, xylose, apiose and myo-inositol (Internal Standard, IS), as well as pyridine, hexane and methanol/3-M HCl solution, were purchased from Sigma-Aldrich. Then, the saccharides were washed with methanol and dried under nitrogen gas flow. Third, the trimethylsilyl reaction was accomplished with 200 µL of Tri-Sil HTP (Thermo Fisher Scientific, Franklin, MA, USA). Each vial with the sample was heated at 80 °C for 1 h. The derivatized sample was cooled to a room temperature and dried under a steam of nitrogen. Forth, the dry residue was extracted with hexane (2 mL) and centrifuged. Finally, the hexane 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.

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 (link)]. 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 (link)], 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.