approximate acyl chain composition (3), other critical information such as the exact regioisomerism of fatty acids along the glycerol backbone, the position(s) of the acyl chain double bonds, the stereochemistry of the double bonds, and so forth, cannot currently be obtained using conventional bioanalytical techniques. For example, a common method of TAG analysis is the chromatographic detection of FFAs or their derived methyl or ethyl esters, which are obtained by hydrolysis from TAGs (4). Although convenient, this method does not allow for consideration of the structural aspects of the intact TAG molecular species (i.e., regioisomeric structural information). This information is critical to understand the dynamic functions of TAGs, their digestion, and their metabolism. A conventional TAG characterization method in MS is based on low-energy (5, 6) or high-energy collision-induced dissociation (CID) (7,8). These methods provide TAG structural information, including acyl chain lengths and double bond number; however, the double bond positions and the acyl chain regioisomeric arrangement are not easily defined due to the limited information derived from the resultant fragments. HPLC has also been used to resolve regioisomers of TAGs (9), but it requires long analysis times, authentic standards as references, and extensive method development, potentially making this method unattractive to a high-throughput lab. A recent study of TAG regioisomers was reported using differential mobility spectrometry (DMS) without HPLC (10). Despite advances in the goal to characterize lipid molecular species, including using ozone-induced dissociation (OzID) (11, 12) or a Paternò-Büchi reaction (13, 14) to determine double bond positions, an efficient method to characterize lipid structural details has been lacking. Recently, a study using OzID found that TAG molecular species containing C18:1, n-7 versus C18:1, n-9 correlated with clinical variables of dyslipidemia and are proinflammatory (15). These results highlight the need for a method to fully characterize Abstract Electron-induced dissociation or electron impact excitation of ions from organics (EIEIO) was applied to triacylglycerols (TAGs) for in-depth molecular structure analysis using MS. In EIEIO, energetic electrons (10 eV) fragmented TAG ions to allow for regioisomeric assignment of identified acyl groups at the sn-2 or sn-1/3 positions of the glycerol backbone. In addition, carbon-carbon double bond locations within the acyl chains could also be assigned by EIEIO. Triacylglycerols (TAGs) are among the most abundant lipids in the human body, located primarily within adipose. TAGs are also found in plants and are concentrated in seeds as a source of energy during embryonic development. It is from these various seeds that a majority of edible oils are derived. The physiological role of TAGs is to serve as a reserve of fatty acids for energy generation via -oxidation. However, these same stored fatty acids may also serve as precursors, either directly or indirectly, to ...