Positional analysis of phosphatidylcholine and phosphatidylethanolamine via LC with a charged aerosol detector

Abstract

A new method for the positional analysis of egg yolk phospholipids (PLs) (phosphatidylcholine-PC, phosphatidylethanolamine-PE) using liquid chromatography with charge aerosol detector (CAD) is described. The method is based on six-step procedure: 1) extraction of phospholipids from tissue sample, 2) separation of lipid classes by solid phase extraction (SPE), 3) complete regiospecific hydrolysis of phospholipids by phospholipase A2 (PLA2), 4) separation of reaction products (fatty acids from sn-2 position and 1-acyl lysophospholipids) by SPE, 5) chemical hydrolysis of 1-acyl lysophospholipids, and 6) analysis of obtained fatty acids by LC with charge aerosol detection (CAD). Total time of enzymatic hydrolysis of PLs ranged from 10-30min. The reaction products were separated by SPE in three-step gradient elution procedure. Chloroform: methanol mixtures were used as eluents to obtain pure fractions of FAs from sn-2 position of PL and 1-acyl lysoPL (chemically hydrolyzed to FAs). FAs were separated by reversed-phase LC using a gradient elution and detected using CAD detector. This combination enables determination of all fatty acids in a single analysis, and without the sample derivatization. The method was optimized and the response of CAD, linearity, precision and sensitivity of the method were studied.

Keywords: Charged aerosol detector (CAD); Fatty acids; HPLC; Phospholipids; Positional analysis; Regiospecific hydrolysis; SPE.