QUANTITATION OF THE FATTY-ACID COMPOSITION OF PHOSPHATIDIC-ACID BY CAPILLARY GAS-CHROMATOGRAPHY ELECTRON-CAPTURE DETECTION WITH PICOMOLE SENSITIVITY

We describe a relatively simple and sensitive method to measure femtom ole amounts of phosphatidic acid in cells. Phosphatidic acid was extra cted from cells in the presence of ptadecanoyl-2-heptadecanoyl-sn-glyc ero-3-phosphate as an internal standard, purified by two-dimensional t hin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluoroben zyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by g as chromatography with electron-capture detection. Long-chain fatty ac ids were resolved with excellent signal-to-noise ratios. Using heptade canoic acid as an internal standard for quantitation, as little as 1 f mol of pentafluorobenzyl ester of stearic acid was detected with a lin ear response up to 10 pmol. Linear detector responses were obtained fo r all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold i ncrease in sensitivity compared to the most sensitive of the previousl y described methods. An example is provided of quantitating phosphatid ic acid from minute amounts of biological samples such as islets of La ngerhans.