Semipreparative isolation of dehydrodiferulic and dehydrotriferulic acids as standard substances from maize bran

A method for the isolation of diferulic and triferulic acids in quantities and purity that comply with the requirements for their use as standard substances was developed. The procedure includes alkaline hydrolysis of destarched maize bran and ether extraction of liberated phenolic compounds. Following a first purification by liquid-liquid extraction Sephadex LH-20 chromatography is performed. This step is the core of the method and allows the separation of monomeric and dimeric/trimeric substances. A good pre-separation of di- and triferulic acids (purity in most cases >75%) is also achieved. Further separation and purification is carried out by semipreparative RP18-HPLC. Using this rapid, easy to handle, and moderately priced separation procedure it is possible to obtain approx. 41 mg 8-O-4'-diferulic acid, 27 mg 5-5'-diferulic acid, 12 mg 8-5'-diferulic acid (benzofuran form), 16 mg 8-5'-diferulic acid (open form), 11 mg 8-5'-diferulic acid (decarboxylated form), 7 mg 8-8'-diferulic acid (cyclic form), 5 mg 8-8'-diferulic acid (open form), and 10 mg 5-6,8'-O-4'-triferulic acid out of 20 g destarched maize bran. The incorporation of minor modifications allows a further upscaling of this procedure.