Determination of prostaglandins, and other metabolites of arachidonic acid by thermospray HPLC/MS using post column derivatization.

Thermospray HPLC/MS analysis of the metabolites of arachidonic acid proved to be sensitive and specific. The compounds included were hydroxy-fatty acids (5-HETE, 12-HETE, and 15-HETE) and prostaglandins PGD2, PGE2, PGF2 alpha, PGA2, PGA1, TXB2, and 6-Keto-PGF1 alpha. Thermospray HPLC/MS analysis allows for simultaneous monitoring of each compound without the need for additional sample preparation or derivatization. The thermospray spectra for the metabolites exhibited [M + NH4]+ ions and fragment ions because of sequential loss of equivalents of H2O. HPLC/MS showed detection limits in the 0.5 to 5 ng range when using multiple ion detection for most of the metabolites. Post-column derivatization of these metabolites using trimethylanilinium hydroxide (TMAH) to form the methyl esters is also presented. This derivatization resulted in a gain in ion current by a factor of 3-6 for most compounds while adding potential specificity to the analysis. The thermospray spectra of the derivatives were nearly identical to the spectra of the free acid except the peaks were incremented by 14 daltons due to the methyl ester formation. The derivatization of the carboxylic acid group proved to be complete under thermospray conditions producing the maximum ion current and causing no compromise in operation of the interface.