Two-dimensional LC-MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver.

A two-dimensional (2D) hydrophilic interaction liquid chromatography (HILIC) and reverse-phase (RP) liquid chromatography (LC) system coupled with triple-quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphatidylcholine in extracted biological samples. Briefly, the 2D HILIC-RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C18 column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however, the absolute matrix effect factor was reduced in 2D LC-MS/MS system. Limits of detection of 2D LC-MS/MS system were 2- to 3-fold lower compared with one-dimensional RPLC-MS/MS. The recovery from the sample ranged from 84.5 to 110%. To summarize, the developed method was proven to be accurate and producible, as relative standard deviations remained <20% at three spiked levels. The efficiency of this newly developed system was applied to measure changes of lipids in the liver of mice after naphthalene treatment. Orthogonal projection to latent structures-discriminant analysis discriminated the lipids from control and the treatment group. We concluded that 2D LC-MS/MS is a promising method to assist lipidomic studies of complex biological samples.