Detection of tobacco consumers using 4-hydroxycoumarin as chromogenic agent in Konig’s reaction

The purpose of this study was to develop and standardize a protocol for the novel use of a benzopyrone derivative, namely 4-hydroxycoumarin (4-HC), for the estimation of cotinine to identify tobacco consumers using Konig’s reaction. 4-HC was soluble in 2-propanol and 1-butanol at a concentration of 100 mg/mL and 80 mg/mL, respectively. Cotinine was estimated by Konig’s reaction using three different formats in which the physical nature of primarily two reagents, potassium thiocyanate and Chloramine-T, was varied. The three formats included dry format, semi-dry format and liqiuid format. The use of 4-HC disssolved in 2-propanol was found to quantitatively estimate cotinine at a lower detection limit of 50 μM in all three formats at 500 nm. A quantitative lower limit of detection of 3.2 μM of cotinine was obtained when 4-HC was dissolved in 1-butanol in semi-dry format wherein potassium thiocyanate and Chloramine-T were used in a combination of liquid and solid form, respectively, at Abs (525 nm)-Abs (750 nm). Besides, quantitative detection, use of 4-HC in 1-butanol results in a biphasic reaction which credits its use for cotinine estimation in a qualitative visual format with the development of a pink polymethine product in the upper 1-butanol layer against the colorless lower aqeous layer.