Quantitation of 1-Stearoyl-2-arachidonoyl-sn-3-glycerol in Human Basophils via Gas Chromatography - Negative Ion Chemical Ionization Mass Spectrometry

Investigation of the role of diacylglycerol molecular species in signal transduction in human basophils has been impeded by the lack of an assay method with adequate sensitivity and selectivity. Conversion of 1-stearoyl-2-arachidonoyl-sn-3-glycerol to the pentafluorobenzoyl ester conveys electron-capture properties to the diacylglycerol. The electron-capture derivative of the diacylglycerol is amenable to gas Chromatographic analysis and undergoes limited fragmentation under negative ion mass spectrometric conditions with generation of an intense molecular anion at m/z 838. Monitoring mlz 838 for detection of 1-stearoyl-2-arachidonoyl-sn-3-glycerol and mlz 841 for detection of 1-trideuterostearoyl-3-arachidonoyl-sn-2-glycerol employed as the internal standard provides the analytical basis for GC-MS quantitation of the endogenous diacylglycerol in human basophils. The assay displays excellent reproducibility over a wide range of concentrations with variations ≤ 10%. The GC-MS assay is highly selective and exquisitely sensitive with a detection limit of ≤0.20 pg (∼30 fmol) for endogenous 1-stearoyl-2-arachidonoyl-sn-3-glycerol per injection. Approximately 400 fmol of the diacylglycerol were extracted from 10⁵ stimulated human basophils.