Optimization for quantification of sorghum tannins by Ferric ammonium citrate assay

Abstract

Due to the complexity of structure, it is difficult to determine the content of sorghum tannins. The current method for the determination of sorghum tannins is the Ferric ammonium citrate assay described in ISO 9648. However, the standard tannic acid (TA) used in the ISO 9648 method is significantly different in both structure and dynamics from sorghum tannins, resulting in inaccurate quantitative result. Furthermore, the extraction solvent, 75% dimethylformamide (DMF), used in each step of this method had large toxicity to human. Hence, the objective of this study is to optimize the ISO 9648 method by selecting proper standard and extraction solvents so as to enhance the accuracy and safety. In this study, Sephadex LH-20 chromatography combined with high pressure liquid chromatography was used to purify and identify the extracted sorghum tannin (ST). Then, commercial TA, epicatechin (EA), grape seed procyanidin (PA) were selected as the standard and ST was used as reference for Ferric ammonium citrate assay to compare the effects of the three standards. Furthermore, the extraction rates of sorghum tannins in the presence of several low toxic solvents, such as absolute ethanol, absolute methanol, 70% ethanol solution, 70% methanol solution and 70% acetone solution, were compared to determine the alternative solvents of 75% DMF solution. The results showed that PA was superior to TA and EA in structure, with satisfactory color yield close to ST, and higher accuracy than TA. In terms of the extraction solvent, 70% acetone solution was selected to replace 75% DMF solution because of its higher extraction yield (only next to 75% DMF) and low toxicity. Verification experiment results showed that both the recovery rate and the repeatability of the optimized method met the requirements of AOCO. Moreover, the optimized method, with higher accuracy and safety than the ISO 9648 method, can be applied widely in laboratory.