Several products of cholesterol oxidation have been found in and isolated from foods and have been reported to have adverse biological activity. The successful separation and quantitation by isocratic HPLC of a wide polarity range of cholesterol oxidation products has eluded chromatographers. This study reports the development of an effective HPLC method for the separation and quantitation of a complex mixture of cholesterol and nine oxidation products by use of a binary solvent system of hexane and 2-propanol in a three-part gradient with detection by an HPLC flame ionization detector. Response factors relative to cholesterol were determined, and coefficients of variation were calculated. Each product gave a linear response (R > 0.99) over a concentration range of 100-0.39 pg/lOO pL.

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A study of the positive and negative ion, ammonia (NH3 and N2H3) direct chemical ionization mass spectrometry of highly purified prostaglandin endoperoxide (PGH2) is presented. The positive ion spectra were characterized by an intense [M + NH4]+ adduct at m/z 370 and several fragment ions, most notably a [M + NH4-H2O]+ ion at m/z 352 and an ion at m/z 298, assigned as the [M + NH4-72]+ ion of 12-hydroxy-5,8,10-heptadecatrienoic acid formed form PGH2 in the spectrometer. The negative ion spectra of PGH2 were characterized by a base peak at m/z 352 [M]- and by an ion at m/z 334 corresponding to the loss of water from the parent ion. A combination of negative ion and deuterated ammonia reagent gas was used in making assignments and in demonstrating that the spectra observed were due to intact PGH2 and its stable PGE2 and PGD2 isomers formed in the spectrometer. In addition, use of the latter reagent gas was shown to clearly distinguish between several arachidonic acid metabolites, differing in their number of exchangeable protons. Furthermore, preliminary results with several stable prostaglandins indicate that the spectra are sensitive to different functional groups that are present. Consequently, it would appear that negative and positive ion, NH3 (N2H3) direct chemical ionization mass spectrometry would be useful in the analysis of labile arachidonic acid metabolites, without the need for prior derivatization.