Policing Polyphenols

When using a coulometric detector with HPLC, analytes can be identified by their retention times as well as their hydrodynamic voltammograms (HDVs). The HDVs are determined by the peak height and peak area over three adjacent sensors, the lower, dominant and area sensors. Antioxidants can in most cases be positively identified by their response across these three channels. Software provided with the detector compares the voltammetric response of an unknown compound to an external standard and assigns two ratio accuracies. The two ratio accuracies compare the response of the unknown to the analyte on the lower to dominant channels and the upper to dominant channels. These ratios provide a numerical indicator of the authenticity and purity of the unknown.

Development of HPLC Procedure Using Coulometric Detection

The USDA researchers developed an HPLC procedure utilizing reverse phase chromatography coupled with a coulometric array detection system for characterizing overall antioxidant capacity and identifying and quantifying individual antioxidants such as in fruits and vegetables. The USDA researchers validated this method by analyzing standard solutions of various antioxidants as well as fruits and vegetables purchased from a local supermarket. They used a Dionex HPLC gradient pump along with the ESA coulometric detection system. Separation was performed on an octadecylsiloxane Hypersil column. Fruits and vegetables were weighed and homogenized in deionized water using a commercial blender. The identities of some of the peaks were further assessed by spiking samples of interest with the relevant standards and comparing the height or area ratios of the adjacent peaks to the dominant one between a standard and the actual sample. The compounds identified in the samples were quantified by fitting the peak area of the dominant channel to the standard calibration curve.

Figure 1 shows a 12 channel chromatogram of a 31-component standard including flavonoids and other antioxidants that shows both retention time and oxidation potential as measured by the coulometric detector. The reproducibility of the method was evaluated by repeatedly injecting the standard solution. The within-day retention time variation for each individual standard ranged from 0.09 to 1.38 percent coefficient of variation (CV). The within-day and between-day voltammetric response variability were tested by repeated injections of the standard mixture. The coefficient of variation of within-day responses ranged from 3.08 percent to 13.46 percent for eight replicates injected on a single day while the CV of between-day response ranged from 4.38 to 17.74 percent for a single injection on each of 10 days. However, the between-day voltammetric response for genistein, quercetin, and pelargonidin #2 were higher which may be due to their low solubility. The detection limit was 20 pg for all components except quercetin which was 1 ug. All compounds except for quercetin, 4-hydroxycoumarin, and gallocatechin gallate gave a linear response at concentrations from 1 to 2000 ng/mL. Hydroxycoumarin and gallocatechin gallate both gave a linear response at concentrations from 1 to 1000 ng/mL. Quercetin produced a linear response at concentrations from 50 to 1000 ng/mL.

The voltammetric response was also measured for 24 fruit and vegetable extracts as shown in Table 2. Each fruit and vegetable had a unique chromatographic and voltammetric peak distribution. The electrochemical data obtained with HPLC and coulometric detection correlated well with the automated oxygen radical absorbance capacity (ORACROO) assay with a peroxyl radical generator. The concentrations of some electroactive compounds were identified in the aqueous extracts of the fruits and vegetables as shown in Figure 2 and Figure 3. For example, catechin, rutin, naringin, vitamin C, and glutathione were detected in strawberries. Positive detection was accomplished by matching retention times and peak purity with the voltammetric response of the standard samples. Many other compounds were unidentified but well separated in fruit and vegetable extracts. The resulting chromatograms provide a fingerprint that characterizes the overall antioxidant status of these fruits and vegetables.