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Heterogeneous Ganglioside Standards in LC-MS/MS: Sensitive Method for Quantifying the Major Molecular Components in Mono-Sialo Ganglioside Standards

Ashta Lakshmi Prasad Gobburi, Renliang Zhang, Belinda Willard, Denise Inman and David J Anderson

A commercially-available mono-sialo (GM1) ganglioside standard consists of three major components with different ceramide structures: C18:0 fatty acid/C18-sphingosine, C18:0 fatty acid/C20-sphingosine and C20:0 fatty acid/C18-sphingosine. The usual multiple reaction monitoring (MRM) liquid chromatography-tandem mass spectrometry (LC-MS/MS) of gangliosides, which monitors the dehydrated sialic acid fragment (m/z 290), cannot differentiate between the individual iso-molecular weight components C18:0 fatty acid/C20-sphingosine and C20:0 fatty acid/C18-sphingosine. Present characterization of ganglioside standards quantifies only the fatty acid content by gas chromatography-flame ionization detection (GC-FID) analysis of the ganglioside mixture and does not parse out the percentages of the individual mono-sialo ganglioside components. In the present work analyzing a heterogeneous GM1 standard, results from a dehydrated sialic acid daughter ion MRM LC-MS/MS determination employing hydrophilic interaction liquid chromatography were combined with results of the fatty acid content determined by GC-FID analysis to sensitively quantify the three predominant individual molecular GM1 components in standards at concentrations as low as 50 ng/mL (Method 1). These dehydrated sialic acid MRM results (Method 1) were confirmed by a less sensitive fatty acid daughter ion MRM LC-MS/MS technique (Method 2) which could only determine molecular GM1 components in standards at high concentrations (1 μg/mL-10 μg/mL). Method 2, however, has the advantage of directly quantifying the three predominant individual molecular GM1 components for comparison with Method 1 results. Equations are derived which incorporate the combined data (Method 1) to calculate percentages of individual mono-sialo gangliosides in the standard. Percentages for the individual mono-sialo gangliosides in the standard differed by at most 2% (absolute difference in the percentages) in comparing the results obtained by the two methods.