Analysis of molecular species of 2H-labelled phosphatidylcholines by liquid-gel chromatography and gas chromatography-mass spectrometry

Abstract

A procedure has been developed for the study of deuterium incorporation into fatty acid and glycerol moieties of individual molecular species of phosphatidylcholines. The phosphatidylcholines are isolated by chromatography on a lipophilic Sephadex anion exchanger and are hydrolyzed with phospholipase C. The 1,2-diglycerides are separated as trimethylsilyl ethers by reversed-phase chromatography on hydroxyalkoxypropyi Sephadex. The deuterium excess of the glycerol and fatty acid moieties of the separated trimethylsilyl ethers is determined by computerized gas chromatography-mass spectrometry using repetitive magnetic scanning of whole spectra or repetitive accelerating voltage scanning of a limited mass range. The trimethylsilyl ethers are then hydrolyzed and 3-perdeuteriotrimethylsilyl ethers are prepared which are separated into individual molecular species by preparative gas chromatography. The 2-acylsn-glycerol 3-perdeuteriotrimethylsilyl ethers are then obtained by brief lipase digestion and isolation by rapid straight-phase chromatography on hydroxyalkoxypropyl Sephadex. The deuterium excess at different carbons of the glycerol moiety is determined by gas Chromatographic—mass spectrometric analysis of the 2-acyl- sn-glycerol 1-trimethylsily-3-perdeuteriotrimethylsily ethers. The analyses can be carried out with 5–400 μg of individual phosphatidyl-cholines in the original extract. Examples of the analysis of phosphatidylcholines in bile of bile fistula rats given [1,1- 2H 2]ethanol are given.