Quantitation of phosphatidic acid and lysophosphatidic acid molecular species using hydrophilic interaction liquid chromatography coupled to electrospray ionization high resolution mass spectrometry

• An extraction method for phosphatidic acid and lysophosphatidic acid was evaluated.
• A method for chromatographic separation of these phospholipid classes was developed.
• This novel method takes advantage of high resolution hybrid mass spectrometry.
• The developed method was successfully applied to murine brain samples.

A method for a highly selective and sensitive identification and quantitation of lysophosphatidic acid (LPA) and phosphatidic acid (PA) molecular species was developed using hydrophilic interaction liquid chromatography (HILIC) followed by negative-ion electrospray ionization high resolution mass spectrometry.Different extraction methods for the polar LPA and PA species were compared and a modified Bligh & Dyer extraction by addition of 0.1 M hydrochloric acid resulted in a ≈1.2-fold increase of recovery for the 7 PA and a more than 15-fold increase for the 6 LPA molecular species of a commercially available natural mix compared to conventional Bligh & Dyer extraction. This modified Bligh & Dyer extraction did not show any artifacts resulting from hydrolysis of natural abundant phospholipids.The developed HILIC method is able to separate all PA and LPA species from major polar membrane lipid classes which might have suppressive effects on the minor abundant lipid classes of interest. The elemental compositions of intact lipid species are provided by the high mass resolution of 100,000 and high mass accuracy below 3 ppm of the Orbitrap instrument. Additionally, tandem mass spectra were generated in a parallel data dependent acquisition mode in the linear ion trap to provide structural information at molecular level. Limits of quantitation were identified at 45 fmol on column and the dynamic range reaches 20 pmol on column, covering the range of natural abundance well.By applying the developed method to mouse brain it can be shown that phosphatidic acid contains less unsaturated fatty acids with PA 34:1 and PA 36:1 as the major species. In contrast, for LPA species a high content of polyunsaturated fatty acids (LPA 20:4 and LPA 22:6) was quantified.