Nanoflow liquid chromatography-tandem mass spectrometry for the characterization of intact phosphatidylcholines from soybean, bovine brain, and liver.

Nanoflow liquid chromatography-electrospray ionization tandem-mass spectrometry (nanoLC-ESI-MS-MS) was applied for the characterization of intact phosphatidylcholine (PC) lipid molecules using a homemade reversed phase capillary column with a pulled tip for direct ESI at positive ion mode. Prior to the analytical column, a short capillary trapping column was utilized for on-line pre-concentration via microcross connection. Separation of intact phosphatidylcholines in the nanoflow LC column was carried out using a binary gradient elution method at 300 nL/min. The structures of the eluted PC components were determined by analysis of the typical fragment ions of PC molecules obtained from collision-induced dissociation (CID) after each precursor scan in mass spectrometry. In the current study, nanoflow LC-ESI-MS-MS analysis of PC molecules demonstrated the ability to obtain clear structural information, such as alkyl chain lengths and the degree of unsaturation with a protonated molecule ([M + H]+) and its characteristic fragment ions ([M + H-RCH2COOH]+, [M + H-RCH=C=O]+, and [M + H-184]+). Results from the nanoflow LC-ESI-MS experiment showed the limit of detection at 3.5 fmol for the 14:0/14:0-PC standard. This technique then was applied to intact PC extracts from soybean, bovine brain, and liver without derivatization and resulted in the identification of 28, 25, and 39 phosphatidylcholines, respectively. The LC-MS-MS method has been shown to be useful for the analysis of low concentration PC molecules in biological samples.