Microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids.
For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH(3)OH for 2 min followed by 1 M HCl/CH(3)OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure. Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.
Itonori
Saki
S
Department of Chemistry, Faculty of Liberal Arts and Education, Shiga University, Hiratsu, Otsu, Shiga 520-0862, Japan. itonori@sue.shiga-u.ac.jp
Takahashi
Masato
M
Kitamura
Tomonori
T
Aoki
Kazuhiro
K
Dulaney
John T
JT
Sugita
Mutsumi
M
D000107:Acetylation; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry; D002849:Chromatography, Gas; D005227:Fatty Acids / Q000032:analysis* / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006028:Glycosphingolipids / Q000737:chemistry*; D006868:Hydrolysis; D000432:Methanol / Q000737:chemistry; D008872:Microwaves; D013107:Sphingolipids / Q000032:analysis*; D013997:Time Factors; D014867:Water / Q000032:analysis
D002241:Carbohydrates; D005227:Fatty Acids; D006028:Glycosphingolipids; D013107:Sphingolipids; D014867:Water; D000432:Methanol