Policing Polyphenols

A substantial research effort is being devoted to better understand the protection against various diseases that epidemiological studies have shown to be provided by certain fruits and vegetables. Researchers generally believe that this protection comes from the activity of antioxidant compounds, particularly polyphenols, found in abundance in many fruits and vegetables.

Polyphenols are a group of chemical substances that are characterized by the presence of more than one phenol group per molecule. These compounds are responsible for providing the coloring of many plants. Various polyphenols have been shown in epidemiological studies to prevent cancers of the colon, esophagus, liver, stomach, lung, breast, pancreas and skin. The result is that a considerable amount of attention is now being focused on polyphenols, and particularly the category of polyphenols known as flavonoids which have been demonstrated in a number of studies to be powerful antioxidants.

Antioxidants are believed to help prevent disease by binding with and destroying free radicals, reducing oxidative damage to cells and biochemicals. (For example, oxidation by free radicals is a precursor to cardiovascular disease.) Therefore, it is becoming increasingly important to identify antioxidant compounds in foods, as well as in urine, tissue and plasma samples, in order to develop disease models and improve the design of epidemiological studies.

One of the more challenging aspects of this research is the identification and accurate measurement of these antioxidants amid the many types and varieties of fruits and vegetables where the compounds can be present in different quantities. For example, Dr. Ronald L. Prior of USDA has determined that the total antioxidant activity of a strawberry is 16 times that of a honeydew melon.

The standard approach for the identification of flavonoids combines the separation capabilities of high-performance liquid chromatography (HPLC) with the pinpoint molecular identification capabilities of mass spectroscopy (MS). This approach has been involved in the discovery of many polyphenols and provides the gold standard of positive identification. On other hand, the large number of polyphenols found in many fruits and vegetables and the large universe of potential plants whose antioxidant activity is of interest create the need for a method of quickly screening food, tissue and plasma samples for a large number of potential antioxidants in a minimal amount of time.

Investigation of Coulometric Array Detection

HPLC/MS is not well suited for this task because of the limited ability of HPLC to separate closely related antioxidants and the relatively large amount of time required for MS analysis.

For these reasons, researchers have looked at other detection methods, often focusing on methods that leverage the antioxidant activity of these compounds for purposes of separation and identification. Dr. Prior and his USDA colleagues Changjiang Guo, Guohua Cao, and Emin Sofic investigated the use of coulometric array detection after separation by HPLC.

Coulometric detection provides a complete voltammetric resolution of analytes as a function of their reaction potential, providing a separate capability that greatly enhances HPLC when dealing with antioxidants. Prior uses include the ESA CoulArray coulometric multi-electrode electrohemical detector in combination with HPLC. The CoulArray detector includes 16 independent electrochemical detectors set to different electronic potentials in series. A given compound can be either oxidized or reduced at a specific potential. This makes it possible to detect and quantify extremely small quantities of electrochemically active compounds in a complex matrix.

The CoulArray can simultaneously determine both retention characteristics and electrochemical potential of the compounds eluted from the HPLC column. This makes it possible to detect many peaks through differences in electrochemical potential that would otherwise be hidden because they elute at the same time. In conventional electrochemical cells, only a small proportion of the sample interacts with the electrode. CoulArray detectors are designed so that the entire sample flows through a porous graphite frit in such a way that the entire sample interacts with the electrode. This greatly improves the sensitivity of the measurement and also makes it possible to perform accurate quantitative measurements.