Spectrophotometric assays assessing total phenol and flavonoid content in plant samples are cheaper and faster and thus more accessible methods than analytical chromatography techniques, although these identify compound categories rather than individual compounds. Most methods are used and are published in several varieties and their general (chemically unspecific) nature is often neglected. The aim of the present in vitro study was to compare five frequently used methods in terms of cross reactivity and sensitivity using pure phenolic substances. Test compounds were selected to represent categories of phenolic compounds of special interest in plant stress studies. Examining the classic Folin-Ciocalteu test we found that in addition to phenolics, it was also reactive to ascorbate. Three flavonoid assays were also examined. These are usually applied to quantify (i) flavonols using aluminium-chloride, (ii) flavanons and flavanonols using 2,4-dinitrophenyl-hydrazine or (iii) flavanols using 4-dimethylamino-cinnamaldehyde. We found that all three methods were unaffected by the presence of ascorbate, but only the last one was specific to the group of compounds aimed at. Our results show that cross reactivities to various flavonoid groups should be taken into account when interpreting data from these assays. Methods utilizing the UV absorption of phenolic compounds were also tested and analyses of whole spectra were found more accurate than estimates based on absorptions at single wavelengths.
Keywords: Flavonoid content; UV absorbing leaf pigments; method optimization; total phenol content.