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Abstract
A novel liquid chromatography-mass spectrometry (LC-MS) approach for analysis of oxidized phosphatidylcholines by an Orbitrap Fourier Transform mass spectrometer in positive electrospray ionization (ESI) coupled to hydrophilic interaction liquid chromatography (HILIC) was developed. This method depends on three selectivity criteria for separation and identification: retention time, exact mass at a resolution of 100,000 and collision induced dissociation (CID) fragment spectra in a linear ion trap. The process of chromatography development showed the best separation properties with a silica-based Kinetex column. This type of chromatography was able to separate all major lipid classes expected in mammalian samples, yielding increased sensitivity of oxidized phosphatidylcholines over reversed phase chromatography. Identification of molecular species was achieved by exact mass on intact molecular ions and CID tandem mass spectra containing characteristic fragments. Due to a lack of commercially available standards, method development was performed with copper induced oxidation products of palmitoyl-arachidonoyl-phosphatidylcholine, which resulted in a plethora of lipid species oxidized at the arachidonoyl moiety. Validation of the method was done with copper oxidized human low-density lipoprotein (LDL) prepared by ultracentrifugation. In these LDL samples we could identify 46 oxidized molecular phosphatidylcholine species out of 99 possible candidates.
Highlights
Non-enzymatic cellular oxidation processes are triggered by reactive oxygen species (ROS)such as hydrogen peroxide, superoxide anions or peroxide anions produced in the respiratory chain in mitochondria
The advantage of recently emerging hydrophilic interaction liquid chromatography (HILIC) separation techniques in lipid analysis is the ability to separate phospholipids by their polar head groups rather than by their non-polar fatty acyl moieties [14,15,16], which is important for lipids with multiple functional groups like oxidized phospholipids
One major aim of this study was the development of a method for separation of oxidized phosphatidylcholines from non-oxidized, as well as from other, lipid classes
Summary
Non-enzymatic cellular oxidation processes are triggered by reactive oxygen species (ROS). The advantage of recently emerging hydrophilic interaction liquid chromatography (HILIC) separation techniques in lipid analysis is the ability to separate phospholipids by their polar head groups rather than by their non-polar fatty acyl moieties [14,15,16], which is important for lipids with multiple functional groups like oxidized phospholipids. Another substantial advantage is “pseudo normal phase separation” using aqueous solvent mixtures compatible with the electrospray ionization (ESI) process. A customized in-house developed software solution [23]
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