Comparison of seed phenolic extraction and assay methods

Summary 1. Phenolic acids have been found to play a role in seed persistence traits such as dormancy and resistance to microbial decay, and so accurate methods for their quantification are needed to study the ecological significance of these compounds. 2. The goal of this study was to compare three different spectrophotometric assays and gas chromatography–mass spectrometry (GC/MS) for the extraction and quantification of phenolic compounds in seeds. Total phenolic concentrations in the seeds of wild oat, broadleaf plantain and common lambsquarters were determined according to the methods recommended for the total polyphenol content assay, the orthodihydroxyphenol assay and the Folin–Ciocalteu reagent (FCR) assay, and compared to totals determined through GC/MS. 3. The spectrophotometric methods were found to consistently overestimate the total phenolic concentrations when compared to totals indicated by GC/MS. The total concentrations indicated by GC/MS analysis ranged from as low as 0·005% of the spectrophotometric totals indicated by the total polyphenol content assay to 12% of the totals indicated by the FCR assay. 4. Ferulic acid, p‐coumaric acid and vanillic acid were among the most abundant simple phenolics in the seeds, with ferulic acid accounting for as much as 56% of the total phenolic composition in the hulls of wild oat seeds. 5. Bound phenolics also constituted a large proportion of the total phenolic concentration (up to 98%) although only one method extracted them. 6. The results suggest that to get an accurate determination of the simple phenolic content of seed material, GC/MS analysis, along with the use of calibration curves and internal standards, is needed.