Simultaneous measurement of serum probucol and lipid-soluble antioxidants

A method is described for the simultaneous measurement of probucol, retinol, tocopherols, lycopene, and carotenes by reverse phase high performance liquid chromatography. A high sensitivity was achieved by use of a microbore column and by monitoring the effluent at the optimum wavelengths of each substance with a diode array detector. The detection limits were lycopene 0.5 ng; alpha-carotene, beta-carotene, and retinol 1 ng; probucol 2 ng; alpha-tocopherol and gamma-tocopherol 15 ng. The eluent was acetonitrile-water-tetrahydrofuran 81.3:5.7:13 (v/v/v) and the flow rate was 0.4 ml/min. Quantitation was performed by use of the four internal standards retinol acetate, 2-pentanone bis(3,5-di-tert)mercaptole, alpha-tocopherol acetate, and retinol palmitate, which resemble the respective analytes in structure and/or polarity. In order to attain a reproducible recovery of particularly the carotenes, the total lipid content of the samples had to be controlled by dilution of the sample before extraction. The coefficients of variation for between-day determinations of a serum pool were 3.8% for retinol, 4.5% for probucol, 11.2% for gamma-tocopherol, 4.5% for alpha-tocopherol, 10.4% for lycopene, 8.0% for alpha-carotene, and 7.0% for beta-carotene.