Determination of coumarin and aflatoxin in tobacco and smokeless tobacco products by liquid chromatography tandem mass spectrometry
A simple and selective method for the determination of coumarin has been developed using isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). In this study, a simple ethanol;water (1:1 v/v) extraction with coumarin-5,6,7,8-d4 as the internal standard was used. The mass detection conditions were optimized utilizing three mass transition (MS/MS) pairs. The method exhibits good linearity (R2 ? 0.999) and a wide concentration range (0.1-1000ng/mL) with a lower limit of quantification (LLOQ) near 67 ng/g. The method was validated with three reference products; Kentucky Reference KY3R4F cigarette filler, 1S2 dry snuff and 2S3 moist snuff. While no coumarin was detected in KY3R4F, coumarin was found in both smokeless tobacco products 1S2 and 2S3 (approximately 1100 and 800 ng/g respectively) with relative standard deviations less than 10% for both samples (n=7). An independent LC-MS/MS method was developed for the determination of aflatoxins (B1, B2, G1 and G2). Tobacco product was extracted with methanol:water (80:20 v/v) using a commercially available internal standard (U-[13C17]-AFB1). After dilution with a phosphate buffer solution (PBS), sample clean up was achieved using an immunoaffinity cartridge. This procedure was simplified when only aflatoxin B1 was required using only water. Three mass transitions for each analyte were evaluated. The method exhibits good linearity (R2 ≥ 0.999) over a concentration range of 0.1 to 100ng/mL. In this study, no detectable levels of aflatoxins were found in the reference products (1S2 dry snuff and 2S3 moist snuff). Method accuracy and precision was assessed using laboratory fortified matrix samples with recoveries ranging from 89.7 to 109% across all aflatoxins tested.