Analysis of phospholipids in microalga Nitzschia closterium by UPLC-Q-TOF-MS

Precise structural identification of phospholipids in the microalga Nitzschia closterium has been established using ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) for direct analysis of total lipid extracts. Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode. Phospholipid molecular species identification was based on the characteristic product ions and neutral loss yielded by different phospholipids under ESI-MS/MS mode. The molecular species were confirmed by the carboxylate anions produced by phospholipids in negative mode; the regiospecificity of the two acyl chains was determined from the ratio of sn-1 to sn-2 carboxylate anion abundances. As a result, 18 lipid molecular species were identified for the first time in this microalga, comprising seven phosphatidylcholines (PC), two phosphatidylethanolamines (PE), two phosphatidylinositols (PI), and seven phosphatidylglycerols (PG). Lipid standards of PC, PE, PI, and PG were added to the total lipids as internal standards for semiquantitative analysis, revealing concentrations of phospholipids in this species between 0.09 and 3.37 nmol/mg. This method can produce a full structural profile of intact phospholipid molecular species and can be used for study of the physiological and ecological functions of lipids by monitoring their individual changes over time.