Fig. 2: Evaluation of dynamic ranges for in-depth lipid annotation with MS-DIAL 5.

a Dynamic range and limit of detection (LOD) required to confirm the presence of diagnostic ions for lipid structure elucidation. The x-axis represents the on-column volume of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) and 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (PAPC), while the y-axis shows the peak heights of diagnostic ions from the centroided product ion spectrum obtained from the product ion scanning mode. The response values of important fragment ions were investigated; for instance, “C10 H loss_1 × 10³” and “SN1 18:2_5 × 10²” denote the LOD values of the H-loss fragment ion at the C10 position and the neutral loss (NL) of sn1-18:2 to characterize the sn-position at 1000 and 500 femtomoles (fmol) on-column volumes, respectively. Even in the authentic standard of PAPC, ions related to sn1-20:4 are detected due to chemical impurities or conformational changes during sample preparation. b Relationship between annotation level terminology and lipid description. The cases of PC and SM were described. c Validation of the MS-DIAL 5 environment for lipid structure description based on EAD-MS/MS spectra quality. Dilution series were analyzed three times at each concentration. The representative annotation was determined as follows: if the same lipid name was annotated in at least two of the three replicates, that name was used as the representative annotation. If the annotation results differed across all three replicates, the lipid with the highest score was adopted as the representative. For example, “x10” indicates a dilution 10 times less concentrated than the original, denoted as “x1.” For sphingolipids, green and red circles represent annotations where OH-positions and both OH- and C = C positions are resolved, respectively. For glycerophospholipids, green and red circles indicate annotations of sn-positions and both sn- and C = C-positions, respectively. Blue, orange, and yellow circles represent annotations at the C = C position resolved, molecular species, and species levels, respectively. If the MS/MS spectrum was not assigned to the precursor ion by DDA, a square shape is used. Incorrect annotations are shown as white fills with a border color indicating the source of the misannotation. Source data are provided as a Source Data file.