Molecular species analysis of phosphatidylglycerols by reversed-phase HPLC/ESI-MS

Abstract

A facile method for the determination of molecular species of phosphatidylglycerols (1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerol, PG) is described. Intact PG isolated from spinach leaves and Escherichia coli IAM12119 was converted into bis(3,5-dinitrophenylurethane) derivatives, followed by reversed-phase high-performance liquid chromatography (HPLC) on an ODS column (25 cm x 4.6 mm i.d., 5 μm particle size) using methanol/water/aqueous ammonium hydroxide (95:5:0.1 or 93:7:0.1, v/v/v) as the mobile phase, which gave clear resolution of individual molecular species containing saturated and unsaturated fatty acids. Peaks were detected at 254 nm UV. Reversed-phase HPLC in conjunction with negative electrospray ionization mass spectrometry (ESI-MS) gave a weak [M + 2H - bis-DNPU - RCO]- ion in addition to a prominent pseudomolecular [M - H]- ion, by which individual molecular species could be identified.