HPLC With Electrochemical and Photodiode Array Detection Analysis of Tocopherol Oxidation and Nitration Products in Human Plasma

Advances in analytical methodologies in the isolation, characterization, and quantitation of lipid-soluble antioxidants, such as vitamin E, have evolved since its discovery in 1922 ( 1 ). The primary purpose of vitamin E (tocopherol) and its tocopherol homologs is to act as free radical scavengers to control lipid oxidation in the body ( 2 ). The physiological and biochemical processes lipid-soluble vitamins possess are beneficial in preventing a number of degenerative diseases and conditions such as Alzheimer’s disease and Parkinson’s disease ( 3 – 7 ), cardiovascular disease ( 8 – 10 ), and cancer ( 11 – 13 ). Pharmaceutical, medical, as well as industrial disciplines have been interested in the clinical outcomes of tocopherol studies. Thus, a critical need exists to measure multiple tocopherols and their oxidation products simultaneously in human tissue ( 2 , 14 – 19 ). Our laboratory is interested in studying lipid nitration products in biological matrices primarily as biomarkers to determine the level of oxidative stress and tissue damage. 3-Nitrotyrosine has been determined to be a useful protein biomarker employed to measure oxidative stress based in previous publications ( 20 – 26 ). The most commonly measured nitrated tocopherol compound of interest is 5-NO ₂ -γ-tocopherol ( 2 , 19 , 27 , 28 ). This and other tocopherol congeners of interest are depicted in Fig. 1 . High performance liquid chromatography (HPLC) with combined electrochemical and photodiode array detection (HPLC-ECD-PDA) is a relatively new, versatile and reliable chromatographic technique employed to generate high sensitivity and selectivity, reproducibility, and accuracy in the determination of tocopherol homologs.

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