The dispersive micro-solid phase extraction method for MS-based lipidomics of human breast milk

A simple and rapid microextraction method ensuring high lipidome coverage was developed for liquid chromatography mass spectrometry (LC-MS)-based lipidomics of human breast milk. The dispersive microsolid phase extraction (D-µ-SPE) technique, coupled with the design of experiment (DoE) method, enabled the study of the influence of several conditions (desorption solvent, stationary phase ratio, and sorption and desorption time) on the lipid extraction process of various lipid classes. The D-µ-SPE-based method, which used a mixture of C18 and zirconia-coated silica gel as the sorbent, allowed for the extraction of a wide range lipid classes characterized by different concentration levels. The developed method simplified the extraction procedure for lipidomics without loss of good reproducibility (70% of the MFs had peak volume%RSD <20% for all the tested stationary phases). The highest lipidome coverage was achieved when 100 µL of the human breast milk (HBM) sample was extracted using 27 mg of C18 mixed with 3 mg of zirconia-coated silica gel as the sorbent and methanol:2-propanol: ammonium hydroxide (14:81:5 v/v/v) mixture as the desorption solvent. The sorption and desorption time did not influence the number of extracted molecular features. The advantages of the present method over the traditional SPE and liquid-liquid extraction (LLE) commonly used in lipidomics are the possibility of mixing sorbents with various sorption mechanisms, which ensures high lipidome coverage, and the use of a small number of materials, including the sorbent and organic solvent.