Brilliant Lipidomics Solutions: Sample Preparation, Discovery and Targeted Approaches

Lipid Separation using UHPLC, SFC and a 2-D LC/SFC System with High Resolution Mass Spectrometry

Lipid samples are very complex. A challenge in the global analysis of lipids is the vast number of subspecies and isomers present in biological samples. The presence of molecular species as isomers or isobars are among some of the greatest challenges to overcome. Lipid stereoisomers may have unique roles in biological processes. It is therefore important to be able to separate the structural and stereoisomers which poses a challenging analytical problem. Reverse phase UHPLC separates lipids according to the hydrophobicity of the fatty acyl chains. It is very common to find coeluting isomers which are very difficult to separate with reverse phase chromatography alone. SFC has the advantage of doing lipid class-based separation. The separation is orthogonal to UHPLC. Very polar lipids elute towards the end of the chromatogram. The disadvantage is again lipids belonging to the same class coelute making lipid annotation challenging. The best of both the worlds would be the flexibility to separate either with SFC or LC in the first dimension followed by an orthogonal separation in the second dimension.

In this webinar, we will show the separation of lipids with UHPLC, SFC and the design of a 2-D SFC/LC system for lipid separation.

Presenter: Sheher Mohsin, PhD (Agilent Senior Applications Scientist)

Sheher Mohsin is a senior applications scientist at Agilent Technologies. She received her Ph. D in physical chemistry from the University of Illinois and an MBA from Rockhurst University. She started her career at the US Environmental Protection Agency working on dioxin analysis with high resolution mass spectrometers. She later joined Bayer and worked in the special analysis lab using mass spectrometry to solve problems in synthesis, impurity determination and submission of final product impurity profile to regulatory agencies. Sheher’s current focus is on lipidomics using GC, LC and SFC separations and mass spectrometry. Sheher collaborates with academic and government researchers working on complex problems to come up with innovative, simplified workflows using the latest tools in separation and mass spectrometry.

A Comprehensive, Turnkey Targeted Method for Plasma Lipidome Profiling Across Large Cohorts

The use of advanced lipidomic techniques such as LC-MS/MS to study the human lipidome at the population level has enabled researchers to probe the relationships between disease states and lipid metabolism. Such studies necessarily involve large sample numbers from population cohorts. Capturing a comprehensive picture of the lipidome requires the analysis of several hundred different lipid species, and thus a balance between depth of coverage and sample throughput. Through a collaboration with the laboratory of Peter J Meikle, Ph.D (Baker Heart and Diabetes Institute), we present a targeted LC/TQ-based method (6495C LC/TQ) for the determination of 763 different human lipid species extracted from 10 µL starting volume of plasma and a total analysis time of 16 minutes. We also demonstrate the high performance of the method across large sample groups and provide details from instrument conditions to data processing for easy implementation of the method across other laboratories.

Presenter: Mark Sartain, PhD (Agilent LC/MS Applications Scientist)

Mark has developed a diverse array of targeted and nontargeted mass-spectrometry based metabolomics and lipidomics applications since joining Agilent in 2012, with a focus on complete hardware/software workflows. Mark began utilizing mass spectrometry in his graduate work to characterize glycoproteins, and later established a novel LC/MS strategy to comprehensively profile and identify complex lipids of Mycobacterium tuberculosis in his postdoctoral fellowship at Colorado State University. He continued designing a wide range of MS-based techniques to measure metabolites and lipids for diverse projects as a Research Scientist at the Institute of Systems Biology (Seattle, WA) prior to joining Agilent.

A Novel Solid Phase Sample Preparation Method for Lipidomic Analysis of Plasma Samples

Lipidomic approaches are widely used to investigate the relationship between lipids, health and disease. Conventional sample preparation techniques for extraction of lipids from biological matrices are based on liquid-liquid extraction (LLE). However, these methods are labor intensive and time consuming and present challenges for achieving reproducible selective lipid extraction. We have demonstrated a novel solid phase extraction approach using Bond Elut Lipid Extraction cartridges. Lipids were profiled in plasma using a combination of reversed phase LC-MS/MS and LC-MS with Agilent Lipid Annotator Software. The Bond Elut Lipid Extraction method was compared to traditional LLE methods for lipid class selectivity and coverage (Folch, Bligh-Dyer, Maytash, BUME) and provided equivalent or better results than LLE methods, but with simplified usability and improved reproducibility.

Compared with LLE protocols, the Bond Elut Lipid Extraction method is more robust, saves labor and time, and has potential to be easily automated. The Bond Elut Lipid Extraction procedure requires 30 minutes per batch compared to 60-90 minutes for the typical LLE methods. The Bond Elut Lipid Extraction procedure yields quantitative results with <10% RSD compared to 10-20% RSD for the LLE methods. Combining the Bond Elut Lipid Extraction Method for sample preparation with the two step LC/Q-TOF Acquisition Cycle and subsequent data processing steps provides a robust and integrated workflow for plasma lipidomics. In addition, we have demonstrated the automation of the Bond Elut Lipid Extraction procedure in a 96-well plate based format with the Agilent Bravo Workstation.

Presenter: James Alexander Apffel, Jr. PhD, (Agilent Research Fellow and Associate Vice President)

Dr. Alex Apffel is currently a Senior Research Fellow at Agilent Research Laboratories in the Biological Chemistry Group. His work focuses on application of novel approaches in analytical biochemistry, separations science, microfluidics, automation and mass spectrometry to Systems Biology. After receiving a BS in Chemistry from University of Hawaii, Alex worked with Prof. Harold McNair at Virginia Tech on Multidimensional Chromatographic Analysis (LC-LC, LC-GC, GC-GC), receiving a PhD in Analytical Chemistry in 1981. He then studied with Prof. Roland Frei on a two-year post-doctoral fellowship at the Free University in Amsterdam, The Netherlands investigating ancillary techniques for micro-HPLC systems such as pre- and post-column derivatization, DLI-LC/MS and sample loading/preconcentration systems. Following post-doctoral studies, he joined the R&D group at Hewlett-Packard Waldbronn Analytical Division in Germany, developing an automated Amino Acid Analyzer, “Amino-Quant”. In 1988, he moved to Hewlett-Packard Scientific Instruments Division to work on LC-MS systems such as DLI, Thermospray, Particle Beam and Electrospray LC/MS.. He has more than forty publications and a dozen patents in areas of applied analytical chemistry.

In 1994, he moved to HP Labs, and subsequently Agilent Research Laboratories, where he has lead a number of projects driving Agilent Analytical capabilities into Bioanalytical Application Spaces. His group’s goals have been to research, develop and prototype experimental methodologies, protocols and informatics focused on systems-level proteomics, metabolomics and lipidomics that exploit, extend and expand the capabilities of Agilent Analytical Platforms. Currently his research focuses on the use of Separations and Mass Spectrometry to expand the power of metabolomic and lipidomic analysis, specifically in the areas of sample preparation, high throughput targeted metabolomics methods and analytical approaches to facilitate metabolic flux analysis.