An efficient shotgun lipidomics strategy was established and optimized for fast 
phospholipid profiling of viscera from three fish species: Lateolabrax 
japonicas, Ctenopharyngodon idellus, and Carassius auratus. This strategy relies 
on direct infusion of total lipid extracts into a tandem mass spectrometer 
without additional separation of the individual molecular species. Four classes 
of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine 
(PE), phosphatidylinositol (PI), and phosphatidylserine (PS), were analyzed, and 
at least 81 molecular species of phospholipids were identified, including 34 
species of PC, 24 species of PE, 12 species of PS, and 11 species of PI, in both 
positive- and negative-ion electrospray ionization mode. The results show that 
fish viscera, which are traditionally discarded as fisheries wastes, are 
nutritional in phospholipids with total contents of the four detected 
phospholipid classes ranging from 1.52 to 3.29 mg/g in the three tested fish 
species. Regardless of the tested fish species, PC and PE are the dominant 
phospholipid classes, followed by PI and PS. Furthermore, principal component 
analysis (PCA) was applied to normalize the relative amounts of the identified 
phospholipid species. The results demonstrate that PS 18:0/22:6, PI 18:0/20:4, 
and PI 18:0/20:5 were the main contributors of cumulative value and could be 
used as an indicator for fish species differentiation. This shotgun lipidomics 
method was >10 times faster than traditional methods, because no chromatographic 
separation was needed. The successful application of this strategy paves the way 
for full utilization of traditionally discarded fisheries wastes and provides an 
alternative means for fish species differentiation.