Direct versus sequential analysis of procyanidin- and prodelphinidin-based condensed tannins by the HCl–butanol–acetone–iron assay

Authors: Grabber, John; Zeller, Wayne

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/3/2019
Publication Date: 7/26/2019
Citation: Grabber, J.H., Zeller, W.E. 2019. Direct versus sequential analysis of procyanidin- and prodelphinidin-based condensed tannins by the HCl–butanol–acetone–iron assay. Journal of Agricultural and Food Chemistry. 68,10, 2906-2916. https://doi.org/10.1021/acs.jafc.9b01307.
DOI: https://doi.org/10.1021/acs.jafc.9b01307

Interpretive Summary: Condensed tannins are common chemical components of plants that affect natural and agricultural ecosystems, food palatability and utilization, and human health. When heated in butanol with small amounts of hydrochloric acid (HCl), condensed tannins produce colored anthocyanidin products that can easily be measured in the laboratory with a spectrophotometer. This provides the basis for the commonly used HCl-butanol assay for detecting and quantifying condensed tannins in plant materials. Several years ago we found that adding acetone to HCl-butanol greatly improved anthocyanidin yields and this paper describes our subsequent work to further optimize and evaluate the assay. We ultimately determined that heating acetone-water extracts and residues prepared from tissue in a mixture of 5% HCl, 6.7% water, 50% acetone, 42% butanol and 0.15% ammonium gave the most accurate estimates of condensed tannin levels in forage, woody plant, food, and food by-product samples. We anticipate our improved method will be widely used by animal nutritionists, ecologists, and food scientists to better understand the functional role of condensed tannins in ecosystems and to improve their use in feeds and foods and for promoting human health.

Technical Abstract: In this study, we optimized the HCl-butanol-acetone-iron (HBAI) assay for the analysis of B-linked procyanidin (PC) and prodelphinidin (PD) based condensed tannins (CT) in forage, woody plant, food, and food by-product samples by direct analysis of whole tissue and by sequential analysis of acetone-water extracts and insoluble residues. The resulting yields of cyanidin and a delphinidin were optimized by heating = 0.25 mg/mL of CT standards, 1 mg/mL of tissue and 1-2 mg/mL of acetone-water fractionated tissue for 3 h at 70 °C in medium containing 5% HCl, 6.7% total water, 50% acetone, 42% n-butanol and 0.15% ammonium iron (III) sulfate. Accurate quantitation required CT standards of known purity that were representative of tissues being analyzed. Both analysis methods provided comparable estimates of total CT for many PD-rich samples, but only the sequential analysis gave good recovery and accurate estimates of CT in PC-rich samples.