Optimized extraction of phospholipids and lysophospholipids for nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry

The efficiencies of four different methods for the extraction of phospholipids (PLs) and lysophospholipids (LPLs) from human plasma samples were examined by comparing extraction recovery values using nanoflow liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS). For recovery measurements, six PL and six LPL standards of different head groups were spiked into a human plasma sample, and the peak areas of each individual species after extraction were measured from the chromatograms of the nLC-ESI-MS runs. Recovery was calculated by comparing the peak area of an extracted standard species with that of the same species' spike after extraction of the same plasma sample. For lipid extraction, four different extraction methods were examined: three based on the Folch method with different organic solvents such as CHCl₃, methyl-tert-butyl ether (MTBE), and MTBE/CH₃OH, and one relatively fast method involving CH₃OH only. Evaluations of recovery showed that the modified Folch method with MTBE/CH₃OH proposed in this study was effective for extracting most PL and LPL standards. Then, the four extraction methods were compared with the identified numbers of plasma PLs and LPLs, of which molecular structures can be confirmed by data-dependent, collision-induced dissociation experiments during nLC-ESI-MS-MS. These results demonstrated that the proposed method yielded the identification of 54 LPLs and 66 PLs from a plasma sample, which was the highest identification rate among the four methods.