Determination of azadirachtin and fatty acid methyl esters of Azadirachta indica seeds by HPLC and GLC

Abstract.

A simple and economical method has been developed to estimate the azadirachtin content and fatty acid composition of neem kernels. Neem kernels are crushed and soaked overnight in ethanol. The extract obtained is analysed by HPLC after filtering through a 0.22 µm membrane. The peaks are separated using acetonitrile–water (40:60) 1 mL min^–1 as the mobile phase on an RP-18 column and monitored at 214 nm. For the determination of fatty acid composition, the fatty acids are directly transmethylated in the kernel powder by heating with methanol–acetyl chloride–benzene (20:1:4, v/v) for 1 h in a water bath. The fatty acid methyl esters (FAMEs) obtained are extracted in hexane and analysed using GLC. The separation of the FAMEs is achieved using an RH-Wax column using temperature programming, 170–200 °C at 2 ° min^–1. The peaks are detected using an FID. Both the methods do not require any clean up or defatting of seeds. This results in faster, easier and more economical sample preparation.