Abstract

Robust methodologies for the analysis of fecal material will facilitate the understanding of gut (patho)-physiology and its role in health and disease and will help improve care for individual patients, especially high-risk populations, such as premature infants. Because Lipidomics offers a biologically and analytically attractive approach, we developed a simple, sensitive, and quantitatively precise method for profiling intact lipids in fecal material. The method utilizes two separate, complementary extraction chemistries, dichloromethane (DCM) and a methyl tert-butyl ether/hexafluoroisopropanol (MTBE) mixture, alone or with high pressure cycling. Extracts were assessed by liquid chromatography−high-resolution mass spectrometry-based profiling with all ion higher energy collisional dissociation fragmentation in both positive and negative ionization modes. This approach provides both class-specific and lipid-specific fragments, enhancing lipid characterization. Solvents preferentially extracted lipids based on hydrophobicity. More polar species preferred MTBE; more hydrophobic compounds preferred DCM. Pressure cycling differentially increased the yield of some lipids. The platform enabled analysis of >500 intact lipophilic species with over 300 lipids spanning 6 LIPID MAPS categories identified in the fecal matter from premature infants. No previous report exists that provides these data; thus, this study represents a new paradigm for assessing nutritional health, inflammation, and infectious disease in vulnerable populations.