Measurement of plasma probucol levels by high-performance liquid chromatography

An improved assay for measuring plasma levels of probucol using reversed-phase high-performance liquid chromatography has been developed and used to measure probucol in cholesterol-fed rabbits. The sample was extracted with iso-octane, reconstituted in acetonitrile and was analysed using a Hypersil ODS column with a mobile phase consisting of acetonitrile-water (96:4, v/v). Detection was carried out using ultraviolet absorption at 241 nm. The lower detection limit for plasma probucol was 0.5 micrograms/ml. Inter-assay and intra-assay coefficients of variation were 1.8-4.8% and 1.6-3.3%, respectively. The method was applied to measurements of probucol in plasma from rabbits receiving a diet containing 1% probucol and 2% cholesterol for five weeks. Probucol levels at five weeks were significantly higher in the animals receiving concomitant cholesterol, and were close to the steady-state values predicted from the one week measurements. Also, the plasma probucol levels were positively correlated with total plasma cholesterol (r = 0.87, p < 0.001). The results indicate that bioavailability of probucol is dependent on the lipid content of the diet.