Analysis of blood HETE levels by selected ion monitoring with ricinoleic acid as the internal standard.

Selected ion monitoring of mass fragments of peaks from capillary gas chromatograms permits a sensitive and selective analysis of positional isomers of hydroxy-eicosanoids (as their methyl ester, trimethylsilyl ether derivatives). Because deuterated analogs of these HETEs are not readily available, stable isotope dilution cannot be easily performed. We have developed a method for the quantitation of HETEs using ricinoleic acid (12-hydroxy-oleic acid) as an internal standard. Ricinoleic acid is monitored at m/z 187, while 5-, 12-, and 15-HETEs (those commonly found in biological systems) are monitored at m/z 255, 295 and 225 respectively. The procedure has proven linear for levels from 0.03 to greater than 3.0 nmol HETE per nmol standard. There is however significant "crossover" among the HETEs, requiring preliminary resolution of the various isomers by reverse phase HPLC in samples containing more than one HETE. Recovery of authentic standards added to various blood samples through the extraction, resolution and analyses averaged over 90%. Using this procedure, mean plasma concentrations were found to be less than 0.1 microM for all HETEs tested, while serum levels were approximately 0.3, 0.8 and 0.1 microM for 5-, 12-, and 15-HETE respectively. Serum formed in the presence of 2mM arachidonic acid plus 25 microM A23187, to maximize HETE production, contained an average of 4, 22 and 1 microM 5-, 12- and 15-HETE respectively. Thus while normal human plasma contains sufficient quantities of the various HETEs to produce some of the biological effects attributed to these metabolites, there is potential for an even greater production when sufficient substrate is available.