Evaluation of Improved and Current Vanillin Based Colorimetric Quantification Methods of Triterpenoids

The most common colorimetric quantification method for triterpenoids utilizes vanillin in strongly acidic conditions to form a colored adduct with the analyte, and it has gained popularity because it is fast and easy to perform. Nevertheless, the detection range of this assay is limited and it is susceptible to side reactions, which cause an interference in the reading. Here, we quantify the rate of this interference in common solvents, and we present an alternative method to minimize the interference, which first incubates the analyte solution in perchloric acid at 60°C before the vanillin is added and finally quenched with acetic acid. We also successfully isolated the adduct that formed and confirmed that the color was due to vanillin polymerizing onto the analyte. The improved method had a correlation coefficient of 0.9959, as well as high accuracy and precision, which had a standard deviation of 0.072 mg/mL, and was not affected by minor changes in the conditions making it a robust method. Furthermore, our method had a limit of quantification of 2.37 mg/mL, which can analyze formulations with ease as well as a limit of detection of 0.782 mg/mL. Triterpenoids are quickly emerging as bioactive compounds, and as a result, our work improves upon current methods to quantify triterpenoids.