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

Abstract

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 CHCl(3), methyl-tert-butyl ether (MTBE), and MTBE/CH(3)OH, and one relatively fast method involving CH(3)OH only. Evaluations of recovery showed that the modified Folch method with MTBE/CH(3)OH 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.

A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

“…Over the years some modifi cations to the original solvent system composition have been introduced, tested, and evaluated; although these alterations are not always fully described by the authors, which makes comparison of the lipid content and composition of fl uids in similar pathological conditions somewhat diffi cult. A comparison of the Folch method and the Bligh and Dyer method has shown similar recoveries in the extraction of the predominant phospholipid classes ( 15 ), and similar conclusions were reported in other studies comparing the Folch method with alternative extraction solvent systems (16)(17)(18). Other solvent mixtures that include, for example, hexane ( 19,20 ), butanol ( 18,21 ), ethyl acetate ( 21 ), and more recently tert-butyl methyl ether (TBME) ( 17,(22)(23)(24) are also described in the literature.…”

“…A comparison of the Folch method and the Bligh and Dyer method has shown similar recoveries in the extraction of the predominant phospholipid classes ( 15 ), and similar conclusions were reported in other studies comparing the Folch method with alternative extraction solvent systems (16)(17)(18). Other solvent mixtures that include, for example, hexane ( 19,20 ), butanol ( 18,21 ), ethyl acetate ( 21 ), and more recently tert-butyl methyl ether (TBME) ( 17,(22)(23)(24) are also described in the literature. TBME, which was reported to give comparable results to the Bligh and Dyer method in the profi le of human plasma ( 16 ), has become particularly popular for sphingolipidomic studies and the extraction of lipids in fl uids ( 21,23 ).…”

“…TBME, which was reported to give comparable results to the Bligh and Dyer method in the profi le of human plasma ( 16 ), has become particularly popular for sphingolipidomic studies and the extraction of lipids in fl uids ( 21,23 ). Generally, the alternative solvent systems were not reported to exhibit signifi cant differences in the extraction of predominant lipid classes when compared with the "traditional" solvent systems (15)(16)(17)(18). Rather, the advantage of these extraction systems lies in the avoidance of chlorinated solvents, which are environmentally harmful.…”

A novel stop-flow two-dimensional liquid chromatography–mass spectrometry method for lipid analysis

“…Lipids extraction method was based on modified Folch method with MTBE/MeOH [28]. After addition of 300 L MeOH to 20 L plasma, the mixture was placed at room temperature for about 10 min after 30 s vortexing.…”

Phospholipid Analysis by Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

“…Recently, nanoflow LC-ESI-MS/MS has been shown to be feasible for qualitative and quantitative analysis of PLs from various biological samples such as tissue, urine, and plasma. 8,24,25,26,27 This review deals with the practical use of nanoflow LC-ESI-MS/MS for the characterization of various PLs from biological samples. Discussed topics are extraction of PLs from biological samples, preparation of capillary columns and nLC separation, effects of ionization modifiers on PL detection during ESI, utility of LiPilot, which is a computer algorithm for structural determination of PLs, quantitative analysis of PLs, and characterization of urinary PLs from breast cancer and prostate cancer patients along with plasma PLs from CAD patients.…”