Comprehensive high-performance liquid chromatographic method for the measurements of lipophilic antioxidants in human plasma.

Owing to the increasing interest in the health effects of antioxidant micronutrients on chronic diseases, a robust and rapid HPLC method for simultaneous measurement of coenzyme Q(10) (ubiquinone and ubiquinol), vitamin A (all-trans-retinol), vitamin E (tocopherols and tocotrienols) and carotenoids (lutein, zeaxanthin, beta-cryptoxanthin, lycopene and beta-carotene) was developed. Sample preparation and analytical conditions that would affect solubility and stability of these antioxidants were investigated and optimized. The mobile phase used was made up of acetonitrile, methanol, ethanol and tert-butanol without corrosive additives such as ammonium perchlorate and perchloric acid. Our results show that using two C(18) columns coupled with photodiode array, fluorescence and electrochemical detection, a comprehensive spectrum of 16 lipid-soluble antioxidants in 30 microL of plasma could be separated and quantified within 30 min. The chromatographic run time was about 3-fold faster and the sample size was about 5-fold smaller than when assays were performed separately using existing methods. The present method will be useful for dietary habit studies and for antioxidant status investigations.