Quantitation of hydroperoxy-eicosatetraenoic acids and hydroxy-eicosatetraenoic acids as indicators of lipid peroxidation using gas chromatography-mass spectrometry.

Peroxidation of cellular membrane lipids has been implicated in a wide variety of acute and chronic pathologies. We have developed a method for quantifying lipid peroxidation products using gas chromatography-mass spectrometry (GC-MS) in order to help elucidate the role of lipid peroxidation in such disorders. The method involves analysis of the methyl ester, trimethylsilyl ether derivatives of various hydroxyeicosatetraenoic acids (HETEs). 16-Hydroxy-9,12,14-heneicosatrienoic acid was synthesized for use as an internal standard. The assay involves the following steps: The internal standard is added to the sample and cellular lipids are extracted and trans-esterified. Next, any hydroperoxides are reduced with triphenylphosphine and the samples are subjected to two steps of solid phase extraction. The samples are then hydrogenated and the trimethylsilyl ether derivative of the hydroxyls formed. The derivatized HETEs are analyzed by electron impact GC-MS. 12-HETE, 11-HETE, 9-HETE, and 8-HETE are assayed by monitoring ions at m/z 301, 287, 259, and 271, respectively. Standard curves were constructed for each HETE and were linear over the range 1 to 250 ng; correlation coefficients were typically greater than 0.99. The assay has been applied to the study of autoxidation of lipids in both in vitro and in vivo systems.