Determination of fatty acid composition in phospholipids using Supercritical Fluid Chromatography - Mass Spectrometry

Abstract

Determining fatty acid composition within different lipid classes is a time-consuming process. Generally, normal phase chromatography is used to separate lipids by lipid class, based on differences in polarity. Fractions of this separation are then collected and derivatized into fatty acid methyl esters (FAMES), followed by analysis with gas chromatography and flame ionization detection (GC-FID). The introduction of electrospray ionization (ESI) and other
soft-ionization methods however have eliminated the need for derivatization as the analyte can be ionized in solution. The use of ESI in conjunction with QTOF-MS is now an established method in determining lipid composition.

This study focuses on the use of ultrahigh performance supercritical fluid chromatography (UHPSFC) in combination with quadrupole time of flight mass spectrometry (QTOF-MS) as an alternative to normal phase chromatography. Although UHPSFC is not yet widely used within lipidomics, interest has been shown in its development for a number of reasons. Compatibility with ESI-MS means that samples can be analysed with a single method, providing an advantage over the time-consuming process of normal-phase chromatography and GC-FID. In addition, carbon dioxide serves as a non-toxic mobile phase characterized by a low critical temperature and pressure that provide efficient chromatography.

The goal of this project was to quantify a broad range of fatty acids within as many phospholipid classes as possible in approximately 10 min of analysis time. Approaches for quantification using internal standards and monoisotopic signal correction methods were investigated for four phospholipid subclasses. Attempts were also made to minimize dynamic ionization suppression although further measurements are required in this area. Internal standards were introduced to the make-up fluid in an effort to handle remaining dynamic ionization suppression. Due to mechanical and experimental set-backs, the method has only been validated for two of the four phospholipids of interest. The study therefore includes
considerations for future measurements.

Popular Abstract

Tackling the diversity of Lipids
Lipids are a structurally and functionally diverse group of biomolecules comprised of several classes. They serve in energy storage and membrane structure, as well as functioning as signal substances and precursors for second messengers. Lipidomics is the emerging field which investigates lipid function on the cellular level. Biological samples are analysed to determine which lipid species are present and at what quantities. This quantitative and qualitative
information aids in the understanding of lipid metabolism, which has been found to play a key role in diseases like diabetes and cancer.

Because of the diversity within and between lipid classes, developing methods which can give quick and accurate results has proved tedious. One of the greatest bottlenecks faced is the processing of data, as many steps are involved in the transformation of raw data into accurate and quantitative results. The lipids must first be identified through the method of choice, and then quantified by comparison with lipid species of known concentration.

In this project, a method was developed based on ultrahigh performance supercritical fluid chromatography (UHPSFC) coupled to quadrupole time of flight mass spectrometry (QTOF-MS) for the analysis of a class of lipids known as phospholipids. In particular, the fatty acid acyl chains which phospholipids are composed of will be examined. UHPSFC is used to separate the lipid classes, which generates individual peaks that can be assigned to a particular lipid. After separation with UHPSFC, the lipids are analysed and detected according to their
mass.

In this study, a method was developed and optimized to quantify a number of phospholipids. Due to mechanical set-backs and time constraints, a number of inquiries remain. Additional measurements are required before truly quantitative data can be produced.

• author: Zandelin, Symantha
• supervisor: Katharina Herzog
• organization: Department of Chemistry
• course: KEML16 20182
• year: 2019
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: Analytical chemistry, Ultrahigh performance supercritical fluid chromatography, Lipidomics, Analytisk kemi
• language: English
• id: 8971945
• date added to LUP: 2019-02-26 11:11:19
• date last changed: 2019-02-26 11:11:19