Determination of total alkaloids as nicotine in tobacco by continuous flow analyzer using stable and safer reagents
ISO 15152:2003 describes analysis of total alkaloids as nicotine (TNA) in tobacco using continuous flow analyzer. It is based on the measurement of the colour complex generated by the reaction of cyanogen chloride and nicotine alkaloids. Cyanogen chloride is generated in-situ by reactions involving chloramine-T and potassium cyanide, a known toxic chemical with LD50 of 5 ppm.In this method, TNA is determined by reaction of an aqueous extract of tobacco with sulfanilic acid and cyanogen chloride. The developed colour is measured at 460 nm. Cyanogen chloride is generated in-situ by reaction between a potassium thiocyanate and a solid stabilized chlorine donor reagent. Various parameters i.e. the concentration and pH of reagents, reaction conditions to form a colour complex, buffer solution, size of sample and length of reaction coil were optimized to obtain results equivalent to the ISO method.The method has been validated as per standard validation protocols i.e. limit of detection, limit of quantification, recovery, precision, accuracy and reproducibility. Recoveries of 97% were obtained with linear regression coefficient of 0.9999.The "r" and 'R" studies on the samples have been conducted with nicotine concentration range from 0.5 to 3.3%. Various tobacco grades with different nicotine concentrations were analyzed simultaneously using both methods and maximum difference in the results were ±0.09 units.