An efficient hydrophilic interaction liquid chromatography separation of 7 phospholipid classes based on a diol column

A hydrophilic interaction liquid chromatography (HILIC) – ion trap mass spectrometry method was developed for separation of a wide range of phospholipids. A diol column which is often used with normal phase chromatography was adapted to separate different phospholipid classes in HILIC mode using a mobile phase system consisting of acetonitrile, water, ammonium formate and formic acid. An efficient between-class separation of seven phospholipid classes including phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinostol, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine was successfully achieved within 14 min using a gradient elution which starts with 90% of organic solvent and ends with 70% of organic solvent. 53 mM formic acid (in both organic phase and aqueous phase) and 60 mM ammonium formate (only in aqueous phase) were used as mobile phase modifier. The relatively high amount of ammonium formate was essential to obtain well-shaped peaks of each phospholipid class, especially phosphatidylserines; actually, no negative effect due to ammonium formate was observed for electrospray-mass spectrometry detection in real-life samples. Good chromatographic separation between different lipid classes was obtained (Rs, from 0.73 to 4.97) and well-shaped peaks (tailing factor, from 0.98 to 1.20) were obtained. The developed method was fully validated and the satisfactory performance characteristics such as linearity (R2, 0.990–0.999), retention time stability (RSD < 1%), within day repeatability (RSD, 5–13%), between day variation (RSD, 7–14%) and recoveries (99.6–115.5%) indicated the gradient HILIC method was appropriate for profiling of plasma phospholipids. The method was successfully applied to separate phospholipids extracts from human plasma, mouse plasma and rat plasma.

► An efficient HILIC separation of 7 phospholipid classes. ► The relatively high amount of ammonium formate is beneficial to separation of PLs. ► The possible mechanism of HILIC separation of phospholipids on diol-bonded column. ► IPA (isopropanol) method for extracting phospholipids from plasma.