Authorship:
Dan Blake (1) Sibylle Heidelberger (1) Rachel Webster (2); Karen Smith (2)
(1) SCIEX, Warrington, UK (2) Queen Elizabeth Hospital, Birmingham, UK
| Short Abstract Research into plasma sphingolipids and determining the concentrations of such is of growing importance in the clinical research laboratory, particularly within groups researching Lysosomal Storage Metabolism. Current methods of analysis primarily involve either enzyme activity procedures or derivatization of compounds prior to analysis. Direct analysis of these groups can be complex due to extensive structural homogeneity between individual compounds. We present here a method for a direct multi-compound approach to this analysis, employing modern advances in column technology to produce a rapid and sensitive LC-MS/MS method for these compounds. |
Long Abstract
Introduction
Research into plasma sphingolipids and determining the concentrations of such is of growing importance in the clinical research laboratory, particularly within groups researching Lysosomal Storage Metabolism. Current methods of analysis primarily involve either enzyme activity procedures or derivatization of compounds prior to analysis. Direct analysis of these groups can be complex due to extensive structural homogeneity between individual compounds. We present here a method for a direct multi-compound approach to this analysis, employing modern advances in HPLC column technology to produce a rapid and sensitive LC-MS/MS method for these compounds.
Methods
Plasma Sphingolipids (Glu-Sph, Gal-Sph, Lyso-Gb3, SPC and the internal standard C-17-SPC) were extracted by solid phase extraction using a method modified (to allow inclusion of additional compounds) from Welford et al (1). Chromatography was performed using a short gradient using HILIC chromatography to allow separation of isomeric and isobaric compounds within the group of lipids under investigation. Mass Spectrometry analysis was provided by a SCIEX TripleQuad 6500 LC-MS/MS system operating in Low Mass Positive Turboionspray mode.
Results
Separation of the structurally similar compounds Glu- and Gal-Sph was achieved by finely adjusting chromatographic conditions at the start of the run. Limits of detection for all compounds was shown to be significantly less than 1ng/ml. Linearity for all compounds investigated was shown to cover at least three orders of magnitude.
Conclusions
We have developed a quantitative method for the extraction and direct analysis of Gal-Sph, Glu-Sph, SPC, Lyso-Gb3 and C17-SPC (internal standard). The method is sensitive and robust with good linearity, and represents a significant improvement on established research methods for investigations of lysosomal storage metabolism. Many current methods employ enzyme activity assays to investigate these metabolic pathways, and often in alternative methods for direct analysis, derivatization is a necessity. Our proposed method negates the need to utilize both of these time consuming and technically in depth techniques, with the additional benefits of modern advances in column technology, allowing for structurally identical yet clinically different compounds to be resolved and individually quantified. The end result of our proposed method is to provide a significantly enhanced suite of information from a single injection for research into lysosomal storage metabolism.
References
(1) Welford R W D et al (2014) Plasma Lysosphingomyelin Demonstrates Great Potential as a Diagnostic Biomarker for Niemann-Pick Disease Type C in a Retrospective Study.