Lipidomics requires the accurate annotation of lipids in complex samples to enable determination of their biological relevance. We demonstrate that unintentional in-source fragmentation (ISF, common in lipidomics) generates ions that have identical masses to other lipids. Lysophosphatidylcholines (LPC), for example, generate in-source fragments with the same mass as free fatty acids and lysophosphatidylethanolamines (LPE). The misannotation of in-source fragments as true lipids is particularly insidious in complex matrices since most masses are initially unannotated and comprehensive lipid standards are unavailable. Indeed, we show such LPE/LPC misannotations are incorporated in the data submitted to the NIST interlaboratory comparison exercise. Computer simulations exhaustively identified potential misannotations. The selection of in-source fragments of highly-abundant lipids as features, instead of the correct recognition of trace lipids, can potentially lead to: (i) missing the biologically relevant lipids (i.e., a false negative), and/or; (ii) incorrect assignation of a phenotype to an incorrect lipid (i.e., false positive). When ISF is not eliminated in the negative ion mode, ~40% of the 100 most abundant masses corresponding to unique phospholipids measured in plasma were artifacts from ISF. We show that chromatographic separation and ion intensity considerations assist in distinguishing precursor ions from in-source fragments, suggesting ISF may be especially problematic when complex samples are analyzed via shotgun lipidomics. We also conduct a systematic evaluation of ESI source parameters on a Exactive equipped with a HESI-II source with the objective of obtaining uniformly appropriate source conditions for a wide range of lipids, while, at the same time, reducing in-source fragmentation.

Lipids from different classes sometimes can exhibit the same exact mass upon electrospray ionization; this presents an analytical challenge in lipidomics. In the negative ionization mode, for example, this can occur with phosphatidylcholines (PCs) and phosphatidylserines (PSs), making them indistinguishable in the absence of fragmentation data. PSs are found at low concentrations in biological samples, making MS/MS spectra difficult to obtain. Moreover, while PCs and PSs are distinguishable in the positive mode, PSs do not ionize as well as PCs, and their ionization is suppressed by the PCs. Here, we show that, in the negative ionization mode, substituting protiated LC-MS additives with their deuterated forms provides a way to distinguish PCs and PSs without chemical derivatization. The method described leverages the differential ionization mechanism of PCs and PSs. PCs are ionized via adduction with salts, whereas PSs ionize via hydrogen abstraction. Substituting the salts used for LC-MS with their deuterated form shifts the mass of PCs by the number of deuterium atoms in the salt, while the mass of PSs remains the same. This comparative shift enables their direct differentiation. We demonstrate that the use of deuterated formate shifts the mass of PCs and provides a direct method to distinguish PCs and PSs, even at biologically-relevant low concentrations. The utility of the method was established and validated in the simultaneous analysis of PCs and PSs in lipid extracts from isolated liver mitochondria in two different rat strains. Thirteen low concentration PSs were identified that would otherwise not have been distinguishable from low concentration PCs.