Sugar linkage analysis by permethylation (glycosphingolipid)

 Transfer an aliquot of glycolipid solution to a round bottom screw glass tube with a PTFE screw cap and evaporate to dryness.

 Add 0.2 mL of dimethyl sulfoxide and dissolved into it.

 Flush N₂ stream into the tube.

 Add about 20 mg of powdered sodium hydoride and 0.2 mL of Iodomethane.

 Immediately capped and vigorously shaken at high speed using a vortex mixer at room temperature for 3 min.

 Place the tube on the ice and add 1 mL of chloroform and 7 mL of water.

 Shake with a vortex mixer and then centrifuge at 3,000 rpm.

 Discard water phase and the add 7 mL of water.

 Wash with water 4 times.

 Transfer chloroform phase to another tube and dry up. [Permethylated glycolipid]

 Add 0.3 mL of a mixture of acetic acid, hydrochloric acid, water (8.0:0.5:1.5, v/v).

 Incubate at 80°C for 18 h.

 Evaporate under N₂ stream with several drops of toluene.

 Dry up in a desiccator at least for 1h.

 Add 0.25 mL of 0.01M- sodium hydride and 0.25 mL of 2 % NaBH₄ in 0.01M- sodium hydride and incubate at room temperature overnight.

 Add two or three drops of acetic acid and methanol.

 Evaporate under N₂ stream.

 Add 0.3mL of 10% AcOH in MeOH and dry under N₂ stream (3 times).

 Dry in a desiccator.

 Add 0.25 mL of pyridine and 0.25 mL of acetic anhydride and incubate in a water bath at 100°C for 10 min.

 Add 1 mL of chloroform and 4 mL of water.

 Wash with water 4 times same as above.

 Transfer chloroform phase to another tube and dry up. [Partially methylated alditol acetates]

 Analyze the partially methylated alditol acetates by GC and GC/MS.

 Transfer an aliquot of glycolipid solution to the one-end rounded glass tube and evaporate to dryness.

(For alternative method, see Comment.)

 Add 0.2 mL of 1M-methanolic HCl and seal another end of glass tube. [Caution: burst or leak]

(For alternative method, see Comment.)

 Incubate at 100°C for 3 h in boiling water bath.

(For alternative method, see Comment.)

 After cooling down, add 0.2 mL of n-hexane and vortex for extraction of fatty acid methyl ester.

 Flush centrifuge and remove upper hexane phase.

 Repeat 2 times for extraction of fatty acid methyl ester.

 Add about 20 mg of silver carbonate to lower phase for neutralization.

 Remove the reaction salts and excess silver carbonate by small scale filtration.

 Evaporate to dryness the methanol phase including methyl-glycosides.

 Add 0.5 mL of methanol, 10 μL of pyridine, and 50 μL of acetic anhydride.

 Incubate at room temperature for 30 min and then evaporate to dryness.

 Quickly add about 100 μL of TMS-reagent and incubate at 60°C for 30 min.

 Add 1 mL of chloroform and 4 mL of water.

 Wash with water 4 times same as above.

 Transfer chloroform phase to another tube and dry up.

 Analyze the trimethylsilyl methyl glycosides by GC and GC/MS. [O-TMS derivatives]

 Prepare methyl glycoside containing O-methylated sugar by methanolysis.

 Add 0.5 mL of dry dichloromethane and dissolved into it.

 Place the glass tube at -80°C in a bath of acetone- dry ice.

 Add 0.2 mL of boron tribromide and maintain at -80°C for 30 min.

 Allow to regain 20°C, and keep overnight.

 Remove solvent and excess reagent by evaporation under reduced pressure.

 Re-methanolysis and prepare O-TMS derivatives for analyze by GC.