Acetone Enhances the Direct Analysis of Procyanidin- and Prodelphinidin-Based Condensed Tannins in Lotus Species by the Butanol-HCl-Iron Assay

The butanol-HCl spectrophotometric assay is widely used for quantifying extractable and insoluble condensed tannins (CT, syn proanthocyanidins) in foods, feeds, and foliage of herbaceous and woody plants, but the method underestimates total CT content when applied directly to plant material. To improve CT quantitation, we tested various cosolvents with butanol-HCl and found that acetone increased anthocyanidin yields from two forage Lotus species having contrasting procyanidin and prodelphinidin compositions. A butanol-HCl iron assay run with 50% (v/v) acetone gave linear responses with Lotus CT standards and increased estimates of total CT in Lotus herbage and leaves by up to 32 fold over the conventional method run without acetone. The use of thiolysis to determine the purity of CT standards further improved quantitation. Gel state C-13 and H-1-C-13 HSQC NMR spectra of insoluble residues collected after butanol-HCl assays revealed that acetone increased anthocyanidin yields by facilitating complete solubilization of CT from tissue.