Comparison of simple monophasic versus classical biphasic extraction protocols for comprehensive UHPLC-MS/MS lipidomic analysis of Hela cells

Abstract In this study, two monophasic isopropanol-water mixtures (IPA:H 2 O 75:25 v/v and IPA:H 2 O 90:10 v/v) were compared with traditionally employed biphasic methods of Bligh & Dyer and Matyash et al. as extraction systems for lipidomics analysis in Hela cells. Samples were analyzed by UHPLC-ESI-QTOF-MS/MS in positive and negative mode using sequential window acquisition of all theoretical fragment ion spectra (SWATH) and a relatively new software (MS-DIAL) was employed for the processing of the data which includes detection of peaks, MS/MS spectra deconvolution, identification of detected lipids and alignment of peaks through the analyzed samples. The studied performance parameters such as precision, recoveries of isotopically labeled internal standards and endogenous lipids, number of extracted lipids, and complexity of employed procedure showed that extraction with IPA:H 2 O 90:10 v/v performs similar to the Matyash protocol and better than Bligh & Dyer as well as IPA:H 2 O 75:25 v/v. However, less complex monophasic protocol which is simpler to implement and can be executed in plastic rather than glass, make the monophasic IPA:H 2 O 90:10 v/v protocol an excellent alternative to the classical biphasic protocols for reversed phase LC-MS lipidomics studies.