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

Abstract 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, γ-tocopherol, α-tocopherol, lycopene, α-carotene and β-carotene among others, using UV detection. Additionally, this method allows the simultaneous separation of probucol, an hypocholesterolemic drug with antioxidant properties. Retinol acetate and α-tocopherol acetate were added to samples as internal standards. A NovaPack ODS C18, 150×3.9 mm, 0.4 μm column was used and the flow rate was set constant at 1 m/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 μg/ml, γ-tocopherol 0.137 μg/ml, α-tocopherol 0.906 μg/ml, lycopene 0.022 μg/ml, α-carotene 0.008 μg/ml, β-carotene 0.015 μg/ml and probucol 1.503 μg/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.