Hydrophilic interaction liquid chromatography–mass spectrometry of (lyso)phosphatidic acids, (lyso)phosphatidylserines and other lipid classes

• Optimization of HILIC separation of (lyso)phosphatidylserines, (lyso)phosphatidic acids and other lipids.
• Acidobasic properties of lipids are correlated with their retention behavior.
• 9HILIC columns with different chemistries are compared for the separation of lipid classes.
• PS, LPS, PA and LPA provide good peak shapes on hydride column at pH 4.
• Final method is applied for porcine brain and kidney extracts.

The goal of this work is a systematic optimization of hydrophilic interaction liquid chromatography (HILIC) separation of acidic lipid classes (namely phosphatidic acids—PA, lysophosphatidic acids—LPA, phosphatidylserines—PS and lysophosphatidylserines—LPS) and other lipid classes under mass spectrometry (MS) compatible conditions. The main parameters included in this optimization are the type of stationary phases used in HILIC, pH of the mobile phase, the type and concentration of mobile phase additives. Nine HILIC columns with different chemistries (unmodified silica, modified silica using diol, 2-picolylamine, diethylamine and 1-aminoanthracene and hydride silica) are compared with the emphasis on peak shapes of acidic lipid classes. The optimization of pH is correlated with the theoretical calculation of acidobasic equilibria of studied lipid classes. The final method using the hydride column, pH 4 adjusted by formic acid and the gradient of acetonitrile and 40 mmol/L of aqueous ammonium formate provides good peak shapes for all analyzed lipid classes including acidic lipids. This method is applied for the identification of lipids in real samples of porcine brain and kidney extracts.