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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Dairy Forage Research » Research » Publications at this Location » Publication #402255

Research Project: Improving Forage Genetics and Management in Integrated Dairy Systems for Enhanced Productivity, Efficiency and Resilience, and Decreased Environmental Impact

Location: Dairy Forage Research

Title: Analysis of total condensed tannins by the HCl-butanol-acetone-iron assay

item Grabber, John
item Zeller, Wayne

Submitted to: Association Official Analytical Chemists Midwest Section Program Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 6/13/2023
Publication Date: 6/13/2023
Citation: Grabber, J.H., Zeller, W.E. 2023. Analysis of total condensed tannins by the HCl-butanol-acetone-iron assay. Association Official Analytical Chemists Midwest Section Program Abstracts. Midwest AOAC Meeting, June 2023.

Interpretive Summary:

Technical Abstract: The butanol-HCl-iron assay is widely used for quantifying condensed tannins (CT, syn. proanthocyanidins) in a wide variety of plants, but the method underestimates total CT content when applied directly to tissue. We tested various cosolvents in the assay and found acetone improved CT quantitation for Lotus tissues with contrasting procyanidin (PC) and prodelphinidin (PD) compositions. We then optimized the HCl-butanol-acetone-iron (HBAI) assay for direct analysis of tissue and for sequential analysis of acetone-water extracts and insoluble residues prepared from forage, woody plant, food, and food by-product samples. Yields of anthocyanidins were optimized by heating = 0.25 mg/mL of CT standards, 1 mg/mL of tissue, or 1-2 mg/mL of acetone-water fractioned tissue for 3 h at 70 °C in medium containing 5% conc. HCl, 6.7% total water, 50% acetone, 42% n-butanol and 0.15% ammonium iron (III) sulfate dodecahydrate. Accurate quantitation required CT standards of known purity sourced from the tissue being analyzed. Both methods provided comparable estimates of CT for most PD-rich samples, but only the sequential method gave full recovery and accurate estimates of CT in most PC-rich samples. Following its introduction, the HBAI method has become a standard method for the analysis of CT in plant materials.