The NaNO2HCl assay

Wilson and Hagerman (1990) described an alternative spectrophoto metric procedure to quantify ellagic acid. This method has three advantages over the procedure developed by Bate-Smith (1977): 1) it is not sensitive to gallic acid, which can result in an overestimate of the ellagitannin content, 2) it is more sensitive, and 3) it is more convenient because oxygen does not interfere with the reaction.

To maximize the formation of a red-colored product in the assay developed by Wilson and Hagerman (1990), (the sample containing) ellagic acid is dissolved in pyridine (which needs to be kept in a fume hood) to a volume of 2.1 mL. A volume of 0.1 mL concentrated HCl is added, and the mixture is brought to 30°C. After a volume of 0.1 mL 1% (w/v) NaNO2 is added, the mixture is quickly mixed and the absorbance at 538 nm is read immediately afterwards (A538,t0). A red product that decays over time forms while the sample is incubated. After 36 min. at 30°C the red product reaches its maximum concentration, and the absorbance (A538,t36) is recorded again. The ellagic acid concentration in the sample is a function of the difference between A538,t0 and A538,t36. Glass or quartz cuvettes need to be used, because pyridine will dissolve plastic cuvettes. The assay is also sensitive to residual detergent in the test tubes, so that new glass test tubes need to be used for each assay.

This procedure is based on the formation of the electophile NO+, which can react with an ellagic acid residue (4.2) at two sites, either via a substitution reaction which results in 4.3, or an addition reaction that results in the nitrosyl dienone 4.4. These compounds can decay to form the quinine oxime 4.5, which under alkaline conditions forms the red product 4.6. When related compounds, such as gallic acid, phloroglucinol, hydroxycinnamic acids, and phenol are subjected to this assay, a yellow-brown product is formed, which does not interfere with the spectrophotometric detection of ellagic acid.

Figure 4-1. Formation of a red chromophore (4.6) from the reaction of ellagic acid with the electrophile NO+, based on Wilson and Hagerman (1990).

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