UHPLC-MS/SRM method for quantification of neem metabolites from leaf extracts of Meliaceae family plants

The botanicals (plant extracts) of neem (Azadirachta indica) have enormous potential to influence modern agrochemical research. Neem is a well-studied plant in the Meliaceae family for its limonoid content; other plants have not been explored in detail. It is imperative to know the specific compounds that are responsible for the insecticidal activity of neem, and their absolute concentration in extracts. We have developed an ultrahigh performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC-MS/SRM) assay and validated the same for five neem metabolites. Using this validated method, azadirachtin A (Aza A), nimbin, salanin, azadiradione (azadi) and epoxy or hydroxy-azadiradione (E/H-azadi) were quantified from neem extracts (leaf, bark and seed) and also from leaf extracts of twelve Meliaceae plants. The E/H-azadi used in the assay was purified from neem leaf extract using a UHPLC based method that we have developed, and the amount was estimated based on the azadi concentration. All analytes showed a linear relationship with regression coefficients higher than 0.993, high accuracy (99-111%) and low coefficients of variation (CVs). The limit of quantification (LOQ) for Aza A (156 pg), nimbin (12.5 pg), salanin, azadi and E/H-azadi (62.5 pg) on column was determined. Using this novel methodology, five neem metabolites were quantified with high sensitivity and specificity. E/H-azadi is one of the most abundant metabolites in neem (10% of the leaf extract) and is also found in higher quantities in the leaf extracts of other twelve plant species in the Meliaceae family. This method has excellent applications for quantifying these five neem metabolites with great precision.