Direct infusion and ultra-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis of phospholipid regioisomers

Phospholipids are important components of cell membranes that are linked to several beneficial health effects such as increasing plasma HDL cholesterol levels, improving cognitive abilities and inhibiting growth of colon cancer. The role of phospholipid (PL) regioisomers in all these health effects is, however, largely not studied due to lack of analytical methods.

Electrospray ionization mass spectrometry in negative mode produces structurally informative fragment ions resulting from differential dissociation of fatty acids (FAs) from the sn-1 and sn-2 positions, primarily high-abundance [RCOO]^- ions. The fragment ion ratios obtained with different ratios of regiopure phospholipid reference compounds were used to construct calibration curves, which allow determination of regioisomeric ratios of an unknown sample. The method was developed using both direct infusion mass spectrometry (MS) and ultra-high-performance liquid chromatography and hydrophilic interaction liquid chromatography mass spectrometry (UHPLC-HILIC-MS).

The produced calibration curves have high coefficients of determination (R²  >0.98) and the fragment ion ratios in replicate analyses were very consistent. A test mixture containing 60/40% ratios of all available regioisomer pairs was analyzed to test and validate the functionality of the calibration curves. The results were accurate and reproducible. However, regioisomeric quantification of certain chromatographically overlapping compounds is restricted by the relatively wide window in precursor ion selection of the MS instrument used.

This method establishes a framework for analysis of phospholipid regioisomers. Specific regioisomers can be quantified using the existing data, and method development will continue with improving chromatographic separation and exploring the fragmentation patterns and efficiencies of different PL classes and FA combinations, ultimately to refine this method for routine analysis of natural fats and oils.