Challenges associated with quantification of selected urinary biomarkers of exposure to tobacco products

Tobacco use, of which cigarette smoking is the most common, is a global health concern and is directly linked to over 7 million premature deaths annually. Measurement of the levels of tobacco-related biomarkers in biological matrices reflects human exposure to the chemicals in tobacco products. Nicotine, nicotine metabolites, anatabine, and anabasine are specific to tobacco and nicotine containing products. However, as nicotine and its metabolites are ubiquitous in the environment, background contamination during sample preparation can occur, making the quantification of target analytes challenging. The main purpose of the present study was to examine quality control measures needed in the determination of urinary nicotine, nicotine metabolites, anatabine, and anabasine. Urine samples (n = 75) and NIST standard reference materials SRM 3671 and SRM 3672 were analysed. A one-step extraction procedure using cold acetone was used in this study, which involved no additional clean up. The blank matrices investigated included synthetic urine prepared with HPLC-grade water, synthetic urine prepared with Milli-Q water, and bovine urine. By adopting strategies for minimizing the background levels, very low detection limits for all the target analytes ranging from 0.025 ng/mL for 3-hydroxycotinine to 0.634 ng/mL for nicotine, were achieved. Recoveries ranged between 67% and 118% with RSD values below 20%. Intra-day and inter-day precisions were in the range of 1.1-11.7% and 4.8-25.2%, respectively. The levels of all target analytes were higher in daily smokers than in non-smokers, with the largest difference observed for 3-hydroxycotinine. No difference was observed in the levels of target analytes between individuals who were former smokers, who never smoked or who were exposed to environmental tobacco smoke (ETS), except for total nicotine equivalents (TNE), which was significantly higher in non-smokers exposed to environmental tobacco smoke compared with study participants who never smoked. The results obtained from SRM 3671 and SRM 3672 could inform a potential certification of additional biomarkers of exposure to tobacco products in those standard reference materials.