Analytical Method Development and Validation for the Estimation of Nicotine Content in Various Tobacco Grades by RP-HPLC

A simple, precise, rapid, selective, and economic reversed phase high- performance liquid chromatography (RP-HPLC) method has been developed and validated for the estimation of nicotine content in various tobacco grades in laboratory prepared mixture on a Phenomenex C18 (250×4.6mm i.d) chromatographic column equilibrated with mobile phase containing Methanol/potassium dihydrogen ortho phosphate buffer. Experimental conditions such as organic phase ratio, flow rate, selection of wavelength, etc. were critically studied and the optimum conditions were selected. Efficient chromatographic separation was achieved with mobile phase containing combination of Methanol & potassium dihydrogen ortho phosphate buffer in ratio of 50:50v/v at flow rate of 1.0 ml/min and eluent was monitored at 260 nm. The sample was injected by using a 20μl fixed loop, and the total run time was 6 min. The retention time for Nicotine was 2.797 min. The method was linear in the range of 25μg/ml to 125μg/ml for Nicotine. The proposed method was successfully applied to the analysis of Nicotine in laboratory prepared mixtures. The developed method was validated according to ICH guidelines. Regression coefficient was 0.9996 with asymmetry of NMT 2 and percentage recovery was 99.70%. LOD and LOQ values were 0.41 and 1.23 respectively and method was found to be satisfactory also estimation of nicotine content in various tobacco grades was obtained. This validated HPLC procedure is economic, sensitive, user-friendly & less time consuming than other chromatographic procedures.