Reassessment of Stereochemical Configuration of Natural Phosphatidylglycerols by Chiral-Phase High-Performance Liquid Chromatography and Electrospray Mass Spectrometry

Abstract

Using chiral-phase high-performance liquid chromatography (HPLC) and electrospray ionization–mass spectrometry (ESI/MS), we have redetermined the stereochemical configuration of some natural and synthetic phosphatidylglycerols (PG). For this purpose, the synthetic and natural PG were converted to theirbis-3,5-dinitrophenylurethanes (DNPU), which were separated by HPLC using two columns having chiral phases of opposite configuration, (R)-(+)- and (S)-(−)-1-(1-naphthyl)ethylamine polymers. The molecular species were identified by on-line negative-ion ESI/MS. Absolute configurations of the resolved peaks were assigned by comparison with the elution order of the corresponding 1(3)-monoacyl-sn-glycerol enantiomers asbis-DNPU derivatives on the same column. The results clearly showed that the PG from cabbage leaf lipids and soybean phospholipids consisted of singleR,Sisomers (1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerols), despite the presence of nonstereospecific phospholipase D in the tissues. On the other hand, the PG derived from egg yolk phosphatidylcholine and glycerol by transphosphatidylation with cabbage phospholipase D was a mixture of 45%R,Sisomers (1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerols) and 55%R,Risomers (1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerols). The PG fromEscherichia colilipids was a mixture of 89%R,Sand 11%R,Risomers. The present study demonstrates that chiral-phase HPLC and negative-ion ESI/MS provide direct and unambiguous information about the configuration, identity, and quantity of molecular species in natural and synthetic PG.