UHPLC-ESI-MS/MS analysis of phosphatidylcholine regioisomers with an algorithmic calculation model

Abstract: Despite phospholipids (PL) having a multitude of important biological functions, the effects of different PL regioisomers remain largely unexplored. PL regioisomers have potential to be used as diagnostic markers, for example differentiating cancerous tissue from healthy, highlighting the need for comprehensive analysis methods that are currently lacking. The objective of this research was to create an untargeted PL regioisomer analysis method.

Determining PL regioisomer ratios is based on the differential dissociation of fatty acids (FA) from sn-1 and sn-2 positions, using high abundance FA anion fragments. For example, with phosphatidylcholine (PC) the sn-2 FA is more easily cleaved compared to sn-1 FA, resulting in differences in the fragment ion ratios. Regioisomer ratios can be calculated utilizing mixtures of regiopure reference standards to establish calibration curves. We have created a fragmentation model that predicts fragmentation efficiencies based on the data obtained with reference standards. The model takes into account the lengths of the acyl carbon chains and the numbers of their double bonds, extrapolating this data for PL compounds with different FA combinations.

To demonstrate the accuracy of the calculation model, we have analyzed PC regioisomers in bovine milk using both authentic calibration curves and the calculation model. A total of 21 PC regioisomer pairs were identified. The results with the two calculation methods for PC regioisomer ratios are in good agreement with an average difference of 3.9 percentage points and a maximum difference of 14.9 percentage points. The results show that unsaturated FAs in bovine milk PCs are more concentrated in the sn-2 position when paired with saturated FAs. When both FAs were unsaturated, no clear positional preference was observed.

The developed method provides a novel approach for accurate analysis of a wide range of PC regioisomers and can be further used in studying their biological and nutritional significance.