Gas-liquid chromatography of N- and O-acylated neuraminic acids

Abstract

The qualitative and quantitative analysis by gas-liquid chromatography of natural and synthetic sialic acids with special reference to O-acylated sialic acids is described. Sialic acids are derivatized by N-trimethylsilimidazol. All hydroxyl groups including the carboxyl group are trimethylsilylated as was shown by mass spectrometry. No elimination of the O-acyl groups occurs during the silylation reaction. The TMS-derivatives of the sialic acids are stable in the refrigerator. The TMS-sialic acids including the isomeric N-acyl- O-acylneuraminic acids can be separated from each other on OV-17 or OV-22. Single peaks having individual retention indices from the following sialic acids were obtained: N-acetylneuraminic acid, N-glycolylneuraminic acid, N-fluoroacetylneuraminic acid and N-chloroacetylneuraminic acid; neuraminic acid-β-methylglycoside or its methylester and N-bromoacetylneuraminic acid-β-methylglycoside; N-acetyl-4- O-acetylneuraminic acid, N-acetyl-7- O-acetylneuraminic acid and N-acetyl-9- O-acetylneuraminic acid; N-acetyl-7,9-di- O-acetylneuraminic acid; N-acetyl-4- O-glycolylneuraminic acid, N-acetyl-9(?)- O-glycolylneuraminic acid and N-acetyl-7(?)- O-glycolylneuraminic acid; N-glycolyl-4- O-acetylneuraminic acid, N-glycolyl-9- O-acetylneuraminic acid and N-glycolyl-7(?)- O-acetylneuraminic acid. On OV-1 N-acetylneuraminic acid and N-glycolylneuraminic acid can be separated from each other and from N-acyl- O-acylneuraminic acids: however, the O-acylated sialic acids elute essentially together from this material. The relative detector responses for the different sialic acids vary, as was shown for N-acetylneuraminic acid, N-glycolylneuraminic acid and N-acetyl-9- O-acetylneuraminic acid. 35% of the radioactivity from radioactive N-acetylneuraminic acid and N-glycolylneuraminic acid eluted correspondingly with the sialic acid peaks detected by the FID. Gas-liquid chromatography enables a sensitive qualitative and quantitative analysis of different sialic acids occurring in biological materials, produced during chemical synthesis or in enzymic assays.