Simultaneous quantification of 4-hydroxy coumarin, coumarin, 6-methyl coumarin and eugenol in cigarette filler and in mainstream smoke using HPLC-PDA
Eugenol is the main volatile compound of extracted oil from clove bud (S. aromaticum L.) that is used in traditional medicine as a bactericide, fungicide and anaesthetic.
Coumarin has long been used by the tobacco industry as an additive in cigarettes. Despite its flavour enhancing properties, numerous studies, beginning in 1855, have indicated that coumarin has toxic effects on the nervous system, heart, blood vessels, and liver of animals as well as inducing cancerous tumours and toxic conditions in humans.
Current literature indicates the GC-MS technique as being suited only for eugenol and coumarin in mainstream smoke and involves tedious headspace solid phase micro-extraction.
Based on these considerations, this study proposes a far simpler analytical process using HPLC with a Photo diode array (PDA) detector. It involves extraction of cigarette or mainstream smoke particulate matter previously collected on a Cambridge filter pad with ethanol. The analytical conditions enable separation of all four compounds eugenol, coumarin, 4-hydroxy coumarin and 6-methyl coumarin using C18 Column [25 cm × 4.6 µm × 5 µm] with water:methanol [50:50] mobile phase and column flow of 0.7 ml/min.
The method has been validated and it exhibits excellent linearity (R2 larger than 0.9998) over a wide range of concentrations [10-1000 ng/ml] and recovery varies from 96 to 102%. The limit of detection is 0.0004 mg/Cig and quantification is 0.0012 mg/Cig. The method is robust in a commercial laboratory setting and can be applied to the above flavoured compounds analysis in different cigarette filler / smoking tobacco products.
This method was recently adopted for the analysis of nine international brand Kretek style samples for the above compounds in cigarette filler and in mainstream smoke.