Sibylle Heidelberger (Presenter)
Sciex
Bio: Sibylle earned an MSc in Biochemistry from the University of Ontario as well as a PhD in Pharmaceutical and Biological Chemistry from the University Of London School Of Pharmacy. She has many years’ experience in biochemistry and joined SCIEX in 2013. Sibylle‘s focus is on Lipidomics application development within the clinical and biochemical research arena.
Authorship: Sibylle Heidelberger (1), Daniel Blake (1)
(1) SCIEX, Warrington, UK
| Short Abstract Research into plasma sphingolipids and determining the concentrations of such is of growing importance in the clinical Research into plasma sphingolipids is of growing importance in the clinical research laboratory, particularly within groups researching Lysosomal Storage Metabolism. Current methods of analysis 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. Differential Ion Mobility Spectrometry offers an additional level of separation based on differences in molecular size, shape, charge state and chemical interactions, rather than mass alone. This allows for simplification of chromatographic and extraction methodologies, enabling the creation of rapid and facile analytical methods for inherently similar analytes. |
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. Differential Ion Mobility Spectrometry offers an additional level of separation based on differences in molecular size, shape, charge state and chemical interactions, rather than mass alone. This allows for simplification of chromatographic and extraction methodologies, enabling the creation of rapid and facile analytical methods for inherently similar analytes.
Methods
Plasma Sphingolipids (Glu-Sph, Gal-Sph, Lyso-Gb3, SPC and the internal standard C-17-SPC) were extracted by a simple protein precipitation / direct injection method. Chromatography was performed using a short gradient on a standard reverse-phase column. Absolute chromatographic separation of the isobaric compounds (Glu-SPH and Gal-SPH) was not necessary due to the use of DMS technology (SelexION® ION Mobility Technology, Sciex). Mass Spectrometry analysis was provided by a TripleQuad 6500 LC-MS/MS system (Sciex) operating in Low Mass Positive Turboionspray mode.
Results
Simple chromatography was achieved on extracted samples with a runtime of 5 minutes. Retention times for all compounds were between 2.6 and 3 minutes. Injection of individual standards of all compounds following optimization of DMS parameters confirmed no interference between isobaric compounds despite no chromatographic separation. Sensitivity was determined by extraction of analyte free matrix and limits of detection were confirmed to be 0.1nmol/L for Lyso-Gb3 and Gal SPH, and 0.5nmol/L for SPC and Glu-SPH. Linearity of response was investigated and curves were shown to be linear up to and beyond 100nmol/L.
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) by LC-DMS-MS/MS. The method employs simple sample preparation (protein precipitation with no sample concentration) and conventional, rapid reversed phase chromatography. The proposed method 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 DMS technology to even further simplify the approach, 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 & Acknowledgements:
The authors acknowledge Dr Rachel Webster, Dr Martin Roch and Dr Karen Smith of the Queen Elizabeth Hospital, Birmingham, UK, for provision of samples for analysis and technical assistance / data regarding alternative methods of lipid analysis.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Sciex |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |