Liquid chromatographic method for the simultaneous determination of different lipid-soluble antioxidants in human plasma and low-density lipoproteins.

We describe a reverse phase HPLC method, employing a simple methanol:water gradient as mobile phase, for the determination of several lipophilic antioxidants, such as retinol, gamma-tocopherol, alpha-tocopherol, lycopene, alpha-carotene and beta-carotene among others, using UV detection. Additionally, this method allows the simultaneous separation of probucol, an hypocholesterolemic drug with antioxidant properties. Retinol acetate and alpha-tocopherol acetate were added to samples as internal standards. A NovaPack ODS C18, 150 x 3.9 mm, 0.4 microm column was used and the flow rate was set constant at 1m/min, which allowed the separation of all the desired antioxidants in a total run time of 35 min. A photodiode array detector was used because of its advantages to study the purity of the peaks, however, any programmable multiwavelength UV/VIS detector could be employed given the good resolution of the peaks. The analytical recoveries of the studied compounds were > 96% and the detection limits were: retinol 0.050 microg/ml, gamma-tocopherol 0.137 microg/ml, alpha-tocopherol 0.906 microg/ml, lycopene 0.022 microg/ml, alpha-carotene 0.008 microg/ml, beta-carotene 0.015 microg/ml and probucol 1.503 microg/ml. The intra- and inter-assay coefficients of variation were calculated by using two human plasma samples with different levels of lipophilic antioxidants. The simplicity, rapidity and economy, make this method suitable for the routine measurement of plasma and low-density lipoproteins antioxidants, and may also be used in large scale epidemiological studies. The method has been used to measure antioxidants in samples from patients undergoing treatment with probucol, showing there is a good correlation between the probucol content in LDL and that in total plasma.