Identification and quantification of molecular species of diacyl glyceryl ether by reversed-phase high-performance liquid chromatography with refractive index detection and mass spectrometry

We have developed a method to identify and quantify the molecular species of diacyl glyceryl ether (DAGE) using high-performance liquid chromatography (HPLC) equipped with a refractive index detector and an electrospray ionization and time of flight mass spectrometer (LC–RI–MS). An octadecyl silica column with a mixture of acetonitrile and dichloromethane (65:35, v/v) as an eluant was used for the HPLC. When the LC–RI–MS method was applied to a mixture of synthetic DAGEs; 1-O-hexadecyl-2,3-dioleoylglycerol (O-16:0–18:1–18:1), 1-O-octadecyl-2,3-dioleoylglycerol (O-18:0–18:1–18:1), 1-O-octadecenyl-2,3-dioleoylglycerol (O-18:1–18:1–18:1), 1-O-octadecyl-2,3-didocodahexaenoylglycerol (O-18:0–22:6–22:6), and 1-O-octadecenyl-2,3-didocosahexaenoylglycerol (O-18:1–22:6–22:6), good separation and quantification were obtained on the refractive index chromatogram. A pseudo-molecular ion [M+NH4]+ and a monoacyl glyceryl ether ion [M−RCO2]+ were observed for all synthetic DAGEs on the mass spectrum. It was found that the fatty acids and glyceryl ether in DAGE could be easily identified by these mass spectra. When this LC–RI–MS method was applied to the DAGEs extracted from muscle of Stromateus stellatus, approximately 18 peaks were observed on LC–RI–MS chromatograms and the major molecular species of DAGEs were identified as O-16:0–18:1–18:1.