Electrospray ionization/mass spectrometry compatible reversed-phase separation of phospholipids: piperidine as a post column modifier for negative ion detection

An electrospray ionization (ESI) compatible separation of phospholipids (PL), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), was performed on a C18 column by reversed phase High Performance Liquid Chromatography (HPLC) with minimal ESI suppression. The mobile phase, used isocratically, consisted of methanol and water. ESI was used to efficiently transfer the ions present in solution to the gas phase for mass spectrometric (MS) detection. Formation of negative ions was reinforced by incorporating piperidine post column. Limits of detection (LOD) and limits of quantitation (LOQ) were experimentally determined to be 20 and 60 fmol/μl, respectively, when acquiring data in the selected ion monitoring (SIM) mode monitoring three ions with a single quadrupole MS. When acquiring data from m/ z 110–900 in the scanning mode, the LOD and LOQ were experimentally determined to be 1 pmol/μl and 3 pmol/μl. When acquiring product ion spectra for m/ z 747, the LOD and LOQ were experimentally determined to be 446 attomol/μl and 1.3 fmol/μl, respectively.