Multivariate calibration for the determination of total azadirachtin-related limonoids and simple terpenoids in neem extracts using vanillin assay

Two-component and multivariate calibration techniques were developed for the simultaneous quantification of total azadirachtin-related limonoids (AZRL) and simple terpenoids (ST) in neem extracts using vanillin assay. A mathematical modeling method was also developed to aid in the analysis of the spectra and to simplify the calculations. The mathematical models were used in a two-component calibration (using azadirachtin and limonene as standards) for samples containing mainly limonoids and terpenoids (such as neem seed kernel extracts). However, for the extracts from other parts of neem, such as neem leaf, a multivariate calibration was necessary to eliminate the possible interference from phenolics and other components in order to obtain the accurate content of AZRL and ST. It was demonstrated that the accuracy of the vanillin assay in predicting the content of azadirachtin in a model mixture containing limonene (25% w/w) can be improved from 50% overestimation to 95% accuracy using the two-component calibration, while predicting the content of limonene with 98% accuracy. Both calibration techniques were applied to estimate the content of AZRL and ST in different parts of the neem plant. The results of this study indicated that the relative content of limonoids was much higher than that of the terpenoids in all parts of the neem plant studied.