Preparation of an acid butanol standard from fresh apples

We have developed a simple method for preparing and verifying suitable standards for the acid butanol assay from a readily available source. Phenolics were extracted from fresh apples with methanol, and sugars were removed from the crude extract by treatment with Amberlite resin before fractionating the proanthocyanidins into ethyl acetate. The ethyl acetate fraction was chromatographed on Toyopearl TSK HW-50F to yield about 50 mg of procyanidin dimer and 35 mg of trimer from 1 kg fresh apple fruit. The purity and identity of the standards was easily confirmed by using ESI-MS. In the acid butanol assay, the pure dimer, trimer and purified Sorghum procyanidin had similar color yields on a mass basis, and produced about three times more color than purified quebracho tannin. This new standard overcomes problems associated with overestimation of tannin due to use of the unreactive quebracho tannin standard. Use of the new standard will enable accurate comparisons of tannin levels between laboratories and will standardize comparisons between species, thus promoting our understanding of the role of condensed tannins in plants.