Mass Spectrometric Methodologies for Analysis of Triacylglycerol and Phospholipid Regioisomers in Natural Fats and Oils

Abstract

Analysis of triacylglycerol (TG) and phospholipid (PL) regioisomers is a challenging task. The liquid chromatographic-mass spectrometric (LC-MS) analysis methods can be divided into two distinct categories: a) direct separation by chromatography or other means such as ion mobility, and b) quantification of regioisomer ratios by structurally informative fragment ions with mass spectrometric methods. Challenges arise from the large number of isobaric lipid species in natural samples, often overlapping chromatographically and sharing structurally informative fragment ions.

Due to long retention times and difficult separation, researchers are moving away from direct chromatographic separation of isomers, using mass spectrometry instead. Many established analytical methods are targeting specific isomers of interest, and high-throughput untargeted analysis remains challenging, even though advancements have been made in the recent years. Fragmentation of glycerolipids is influenced by acyl chain lengths and numbers of double bonds of the attached fatty acids, and while certain fragmentation mechanisms offer better sn-specificity than others, the lack of available regiopure standards is still an obstacle for many researchers.

In the thesis work, new tandem mass spectrometric (MS2) methods for investigating TG and PL regioisomer compositions in natural samples were developed. Two different instrumental methods and algorithmic calculation models were developed and validated for analysis of TG regioisomers. A direct inlet negative ion chemical ionization method along with an updated version of the MSPECTRA calculation software offers a rapid protocol for investigating TG regioisomers in complex samples such as human milk. While the absolute accuracy of the regioisomer ratio calculations is affected by the nature of the FAs in the TGs, the method is very useful for studying regioisomeric differences between samples.

A new fragmentation model for analysis of TG regioisomers utilizing positive electrospray ionization (ESI+) LC-MS2 method was developed in addition to the direct inlet method. The model was created using calibration curves established with a wide range of regiopure TG standards, allowing prediction of fragmentation patterns for TG species without authentic standards. While the triple quadrupole MS instrument used for establishing the model is better suited for targeted approaches, the analysis can later be transferred to an untargeted platform utilizing data-dependent acquisition.

Both calculation programs utilize optimization functions, attempting to find concentrations of regioisomers that produce the experimentally observed fragment ion spectra. This approach mitigates the effects of interfering isobaric fragments resulting from different isomeric TG species and the entire fragment spectra of multiple TG molecular species is handled simultaneously instead of each individually.

The analysis of PL regioisomers was divided into two parts. In the first part, a hydrophilic interaction liquid chromatography (HILIC) method combined with a data-dependent MS2 acquisition was used to study the fragmentation behavior of different PL classes. Calibration curves were created with PL reference standards, enabling regioisomeric analysis of specific PL molecular species. For second part of the PL regioisomer analysis, the HILIC separation method was replaced by reversed phase chromatography, resulting in more reliable and efficient separation of PL molecular species within classes. An automated data preprocessing algorithm and a fragmentation model for untargeted PC regioisomer identification was created.

Together, the developed LC-MS2 methods and calculation software and fragmentation models for TG and PL regioisomer analysis form a strong basis for further development and implementation to lipidomics platforms, enabling more detailed structural analysis in a rapidly growing area of research.