RESOLUTION AND QUANTITATION OF THE PREDOMINANT GEOMETRIC BETA-CAROTENE ISOMERS PRESENT IN HUMAN SERUM USING NORMAL-PHASE HPLC

An isocratic high-performance liquid chromatography (HPLC) method completely resolving all-trans-, 9-cis-, and 13-cis-beta-carotenes in human serum was developed. The chromatographic separation was achieved on a calcium hydroxide stationary phase following sample extraction and fractionation on fully activated neutral alumina. A combination of p-methylanisole and acetone was used to modify the hexane (1:1:98) mobile phase, which improved the tailing of later eluting peaks generally associated with chromatographic separations on calcium hydroxide. Sample fractionation on alumina was used to remove components present in serum that interfered with the HPLC separation of beta-carotene isomers. The average analytical recovery was greater than 90% for all-trans-, 9-cis-, and 13-cis-beta-carotenes, and control studies demonstrated that minimal isomerization occurred during extraction and analysis of serum samples. Analysis of pooled serum samples indicated that 13-cis-beta-carotene was the secondary beta-carotene isomer present in human serum with the all-trans form predominating.