Determination of total alkaloids as nicotine in tobacco by continuous flow analyzer using potassium thiocyanate
ISO 15152:2003 describes analysis of total alkaloids as nicotine (TNA) in tobacco, using continuous flow analyzer. The ISO method is based on measurement of colour of the complex generated in situ through reactions involving potassium cyanide, a known toxic chemical with LD 50 of 5 ppm. The titled method based on potassium thiocyanate (KSCN) has been mentioned in the ISO standard as a possible alternative, however, no details have been given. Potassium thiocyanate has LD 50 of 854 ppm and hence is safer to use, handle and store as compared to potassium cyanide. In the present method, TNA is determined by reacting aqueous extract of tobacco with sulfanilic acid and cyanogen chloride, generated in situ by the reaction between potassium thiocyanate and sodium hypochlorite, to form the colour complex. The developed colour is then measured at 460 nm. All relevant parameters i.e. the concentration of reagents, potassium thiocyanate, sodium hypochlorite, and buffer solution, size of sample tubing and length of reaction coil were optimized to obtain results equivalent with ISO method. The KSCN method has been validated as per standard validation protocols i.e. determination of limit of detection, limit of quantification, recovery, precision, repeatability, reproducibility and ruggedness. Recoveries of 97% were obtained with linear regression coefficient of 0.9999. About 800 samples from different tobacco grades with different nicotine concentrations were analyzed simultaneously using both methods. r and R studies on the samples with Nicotine concentration range 0.5 to 3.3% showed the deviations between the results from the two methods to be ±0.1 units.