Antioxidants attacked

There are established links between the consumption of foods containing high levels of antioxidants and protection against diseases related to oxidative stress, such as diabetes, cancer, stroke and heart disease. As a result, there is great interest in the measurement of antioxidant levels in fruit, vegetables and beverages, as well as human biological fluids.

The better methods separate the antioxidants first before measurements are taken, so that the levels of individual compounds can be determined, rather than simply the total levels of antioxidants present. To this end, several methods based on HPLC have been developed and one of more popular ones involves a free radical scavenger.

A solution containing the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical is added post-column to react with the separated antioxidants, which are radical scavenging species. This reaction depletes the radicals and their purple colour is changed to a pale yellow as the corresponding hydrazine derivative is formed. The antioxidants are measured from the change in absorbance by a UV-visible detector.

There are many different published versions of this assay, with such variations as the size of the DPPH reaction coil, the reaction conditions and the solution flow rate. Now, researchers in Australia have attempted to produce the definitive DPPH method by optimising the experimental factors believed to influence the reaction with antioxidants.

Paul Francis and colleagues from Deakin University, Geelong, and the University of Western Sydney used eight common food-based antioxidants as model compounds, such as caffeic acid, ferulic acid, quercetin and rutin. Mixtures of the analytes were separated on a monolithic silica column with a methanol-citrate buffer as mobile phase.

They optimised the concentration and pH of the DPPH solution and studied the effects of degassing the reagent solution with nitrogen or helium. The length and temperature of the reaction coil were also examined.

The chosen set of conditions was then applied to the analysis of antioxidants in an extract of dried thyme and an infusion of green tea. For thyme, the same compounds were detected as in published studies, but their peaks were significantly more intense. In addition, the shorter reaction coil provided greater chromatographic resolution.

The antioxidants were more concentrated in the tea infusion than in thyme. However, the optimised procedure provided better resolution, displaying four distinct peaks in one region where the published methods displayed two broad peaks with shoulders.

So, by careful examination of the individual factors involved in the reaction of DPPH with the antioxidants, better sensitivity and resolution were achieved, although their detection limits were not published in this report.

A team of Turkish researchers took a different view to the DPPH methods, claiming they have several inherent weaknesses, including the short lifetimes of the DPPH radicals. Their alternative was to apply the CUPRAC reagent, which was developed in their own lab, and has been shown to be less dependent than other post-column reagents on pH, mobile phase composition, dissolved oxygen and daylight.

CUPRAC is the Cupric Reducing Antioxidant Capacity method and uses a Cu(II)-neocuprine reagent for the post-column reduction of antioxidants eluting from the column. The change in absorbance of the reagent at 450 nm is measured for antioxidant quantitation, as the reagent colour is changed from light blue to pale yellow during its reduction to the Cu(I)-neocuprine complex.

Resat Apak and co-researchers from Istanbul University optimised their method using standard solutions of common antioxidants. They were detected at 280 nm after elution from a C18 column with a gradient of methanol in ortho-phosphoric acid, before post-column reaction with CUPRAC in a reaction coil and detection at 450 nm.

The detection limits were about 0.2-3.5 µM, which were up to two orders of magnitude lower than previously reported values for the DPPH assay. The linear calibration ranges were 40-200 µM.

The method was demonstrated with the analyses of the antioxidants in extracts of dried green tea, mint and sweet marjoram. The main antioxidants were identified in each case and their contributions to the total antioxidant content were estimated. Flavonoids, phenolic compounds such as catechins, and hydroxycinnamic acids were found.

The researchers preferred the CUPRAC reagent because it did not suffer some of the problems encountered with DPPH, such as sensitivity to air, light, solvents and complex reaction kinetics. It facilitated the analysis of the antioxidants in plants and should provide a competitive alternative to the existing radical-based assays.

Related links:

* Analytica Chimica Acta 2010 (Article in Press): "High-performance liquid chromatography with post-column 2,2'-diphenyl-1-picrylhydrazyl radical scavenging assay: Methodological considerations and application to complex samples"
* Analytica Chimica Acta 2010, 674, 79-88: "Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection"