Methyl-beta-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence for separation and detection of phospholipids.

Abstract

Micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection was applied to the separation of amino group-containing phospholipids including phosphatidylethanolamine (PE), phosphatidylserine (PS), lysophosphatidylethanolamine (LysoPE), and lysophosphatidylserine (LysoPS). A fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), was successfully used to fluorescently label these phospholipids. 4-Fluoro-7-nitrobenzofurazan only produced fluorescent product from LysoPE and PE; signals were not observed from LysoPS and PS. A borax buffer containing sodium deoxycholate modified with methyl-beta-cyclodextrin (methyl-beta-CD) was an excellent MECC system for these phospholipids. Under the optimum conditions, four FQ-labeled phospholipid classes were separated within 8 min. Moreover, each of the PE, PS, LysoPE and LysoPS peaks split into two components corresponding to subclasses with different lengths of the fatty acid chains, but these subclasses were completely resolved only for LysoPE. Detection limits ranged from 0.18 to 1.1 fg (10(-9) - 10(-10) M), which was four- to five-orders of magnitude superior to previously reported CE methods.