Background: Epidemiologic evidence suggests that the concentrations of antioxidant vitamins in human plasma may play an important role in numerous chronic diseases, such as cancer and cardiovascular disease. However, methods for simultaneous measurement of these antioxidants are scarce. We developed and validated a new HPLC method for simultaneous determination of these vitamers in human plasma that uses a novel column-switching approach.
Methods: The new method uses liquid-liquid extraction and isocratic separation with two monomeric C(18) columns maintained at 35 and 4 degrees C coupled with ultraviolet-visible and fluorometric detection. This method could separate 14 vitamers and 3 internal standards within 27 min. No additional modifier was required; the mobile phase was acetonitrile-methanol (65:35 by volume), and the flow rate was 1 mL/min.
Results: For photodiode array detection, the detection limits (signal-to-noise ratio >3) were 0.02 mg/L for beta-carotene, lutein, zeaxanthin, and canthaxanthin; 0.01 mg/L for all-trans-retinol, beta-cryptoxanthin, alpha-carotene, and lycopene; and 0.1 mg/L for all tocopherols and tocotrienols. The detection limit was at least 25-fold lower (0.004 mg/L) when fluorometry was used for measurement of delta-, gamma-, and alpha-tocotrienol and delta-tocopherol compared with ultraviolet detection. The recovery and imprecision of the assay were generally >90% and <10%, respectively.
Conclusions: This new method separates a wide range of fat-soluble antioxidant vitamins in human plasma, including six carotenoids, three isoforms of tocotrienols and tocopherols (delta-, gamma-, and alpha-), and all-trans-retinol. The overall findings suggest that our method is faster, more sensitive, and more comprehensive than existing methods.