Quantitative stereochemical analysis of subnanogram amounts of 12-hydroxy-(5,8,10,14)-eicosatetraenoic acid by sequential chiral phase liquid chromatography and stable isotope dilution mass spectrometry.

The two enantiomers of 12-hydroxy-(5,8,10,14)-eicosatetraenoic acid (12-HETE) are products of different biosynthetic pathways and have distinct biologic actions. Conventional methods of stereochemical analysis of 12-HETE require multimicrogram amounts of material and cannot be applied to systems where the availability of tissue is limited and only trace quantities of 12-HETE are generated. We have developed a method capable of measuring subnanogram amounts of 12-HETE enantiomers which involves addition of racemic. 18O2-labeled 12-HETE as an internal standard, chiral phase HPLC of the pentafluorobenzyl ester derivative of 12-HETE, and stable isotope dilution gas chromatographic-negative ion chemical ionization mass spectrometric quantitation of the resolved stereoisomers. This method has been employed to determine the stereochemical composition of 12-HETE produced by isolated pancreatic islets.