High pressure liquid chromatography methods for separation of ω- and (ω-1)-hydroxy fatty acids: Their applications to microsomal fatty acid ω-oxidation

Abstract

Fatty acids (C 12C 18) and their ω- and (ω-1)-hydroxy derivatives, when converted to p-bromophenacyl (PBP) esters, can be completely separated from one another by high pressure liquid chromatography (HPLC) on a silicic acid column using 0.5% ( v v ) isopropanol in n-hexane. In this system, fatty acid PBP esters are eluted at the solvent front, whereas the retention times of the ω- and (ω-1)-hydroxy derivatives are 14–20 and 24–29 min, respectively. The PBP esters can also be separated by reverse phase HPLC on a μBondapak C 18 column, a method which has been developed by Fan et al. (Fan, L. L., Masters, B. S. S., and Prough, R. A. (1976) Anal. Biochem. 71, 265–272) for separation of methyl esters of fatty acids and their ω- and (ω-1)-hydroxy derivatives. In the latter method, however, the retention times of ω- and (ω-1)-hydroxy derivatives are only about 2 min apart and an increase in the solvent polarity is needed for elution of the esters of unmodified fatty acids. Fatty acid PBP esters, however, can be obtained as independent peaks which are not disturbed by the solvent front. An application of the former method to measure fatty acid ω oxidation by liver microsomes and by a reconstituted monooxygenase system containing purified cytochrome P-450 is described.