Stop-flow comprehensive two-dimensional liquid chromatography combined with mass spectrometric detection for phospholipid analysis.

A novel comprehensive two-dimensional liquid chromatographic (LC × LC) system for characterization of phospholipid (PL) molecular species belonging to six phospholipid classes was developed. To tackle such a task, a silica hydrophilic interaction liquid chromatography (HILIC) column was used as the first dimension ((1)D), and reversed-phase liquid chromatography (RP-LC) with a C18 column was used as the second dimension ((2)D) in combination with mass spectrometric detection. Fraction transfer from the (1)D to the (2)D was performed by means of a two-position ten-port switching valve, operated under stop-flow conditions. The capability of the investigated LC × LC approach was demonstrated in the separation of phospholipid molecular species contained in two Folch-extracted cow's milk and plasma samples. Within the phosphatidylcholine class, up to 16 and 14 different species were identified, respectively, by means of the HILIC×RP-LC-MS system employed under both positive and negative ionization mode.