MSACL 2019 EU Abstract

Plasma Lysosphingolipid Analysis by LC-Differential Mobility Spectrometry-MS/MS (LC-DMS-MS/MS): Rapid Sample Preparation and Resolution of Stereoisomers

Cyrus Papan (1) Daniel Blake (2) Sibylle Heidelberger (2) Mark Jones (3) Charles Turner (4) Neil Dalton (4)
(1) SCIEX, Darmstadt, Germany, (2) SCIEX, Warrington, UK (3) Guy's Hospital, London, UK, (4) Evelina Children's Hospital, London, UK

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Cyrus Papan (Presenter) SCIEX

Relevant Financial Disclosures (within past 24 months) Salary SCIEX Conflict mitigation NOT REQUIRED.

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. It is thought that plasma lysosphingolipids (LysoSLs) are potentially important biomarkers implicated in a range of sphingolipidoses 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.

Sample Preparation:
Extraction was achieved by protein precipitation/direct injection approach utilizing 20μL of plasma

HPLC Conditions:
Short chromatography was provided by a C8 column and a gradient of acidified acetonitrile/water

MS/MS Conditions:
Sciex Triple Quad 6500+ fitted with SelexION DMS Technology, operating in Positive Low Mass MRM

Results
Ion Mobility can separate the stereoisomers Glu-and Gal-SPH in extracts without the need for chromatographic separation
Ion Mobility has been shown to remove interferences that can cause to misinterpretation of the results
Results and statistics in plasma show good Accuracy (88 – 105%) Precision (1.5 – 19%) and Linearity (>0.997) with sensitivity of all compounds in plasma significantly below 0.1ng/ml.
Results generated using this methodology in research samples show good correlation with current methodologies.

Conclusions
We have presented here a method for the direct analysis of a series of sphingolipids in plasma using a simple protein precipitation, standard chromatography and Differential Ion Mobility Spectrometry. This improved methodology for plasma LysoSL analysis using LC-DMS-MS/MS has a simplified sample preparation stage compared to previous methods and can differentiate between stereoisomers without the need for chromatographic separation. Biomarker analysis could be automated and is particularly useful where sample volume is limited (and insufficient for enzyme analysis) or where more invasive and time-consuming methods are currently employed, e.g. skin biopsy.