Development of a sensitive HPTLC method for quantification of nimbolide in Azadirachta indica and its dosage form.

An improved and sensitive High Performance Thin-Layer Chromatography (HPTLC) method has been developed for determination of anticancer compound, nimbolide in different parts of Azadirachta indica and its dosage form. A quick and simple ultrasonication technique was used for the preparation of sample solutions. Separation of the components was achieved on precoated TLC plates by using optimized tertiary mobile phase consisting of n-hexane:ethyl acetate:acetic acid (6:4:0.2, v/v/v) with a solvent migration distance of 68 mm. Densitometric quantification was performed after derivatization of the plate with methanol-sulfuric acid reagent in reflection/absorption mode at 515 nm. A linear response of nimbolide was obtained over the range of 200-1400 ng/spot with a correlation coefficient of r(2) = 0.99968, indicating good relationship between concentration and peak area. The method sensitivity was found to be increased by performing the analysis after derivatization with methanol-sulfuric acid reagent. The limit of detection and limit of quantification were found to be 70 and 200 ng/spot, respectively. The obtained recovery range from 96.70 to 98.01% with an average of 97.46% proves excellent accuracy of the method. ICH protocol was followed for validation of the HPTLC method in terms of precision, repeatability and accuracy. The developed method was found to be highly sensitive and the mobile phase efficiently separated nimbolide from other components. The maximum content of nimbolide was found in leaves. Further, the developed HPTLC method can be applied successfully for the marker evaluation of the formulations containing A. indica.