Sep pak c 18 reverse phase cartridges

The total ganglioside content in the intestinal mucosa was determined by measuring the GG bound sialic acid with GC-MS. The GG were extracted and purified by using the Sep-Pak C18 reverse-phase cartridges (Waters, Milford, MA) according to the method of Schnabl et al. (2009) . The GG were derivatized by trimethylsilylation. One hundred-microliter ganglioside samples with 5 µg of phenyl N-acetyl-α-d-glucosaminide as internal standard were dried and treated with 700 µL of 0.05 N fresh methanolic HCl by heating for 1 h at 80°C. The mixture was cooled and extracted with 0.5 mL of hexane to remove the liberated fatty acid esters. The methanolic layer was dried under a nitrogen stream. The trimethylsilylation derivatization reagent was formed according to the method of Carter and Gaver (1967) . The silylation reagent (50 µL) was added to the samples in the small ample vials. The samples were vortexed and allowed to stand for 15 min. The deriva-tized samples were transferred into GC inserts and 1 µL was injected per assay into a DB-5 GC Column (Agilent, Santa Clara, CA) installed on a Shimadzu GC (Shimadzu, Colombia, MD) coupled with a MS. The quantification was achieved from the standard curve generated by concurrent analysis of a series of ganglioside GD3 standards in different concentrations.