Used and unused snus and gum samples were analyzed for nicotine, which was expressed selleck chem Cabozantinib on a whole portion, wet-weight basis. The analysis of the snus samples was performed at British American Tobacco (Investments) Ltd., Group R&D, Southampton, United Kingdom by GC-MS. The nicotine gum samples were analyzed at Eurofins Food & Agro Sweden AB, Lidkoping, Sweden by LC-MS. Sensory Evaluation While using the administered snus products, subjects were requested to complete a short sensory questionnaire, consisting of four questions, to assess their sensory experiences relating to irritation of lips and throat, level of salivation, or other perceived sensations such as any ��buzz�� feeling during use of the snus. Subjects scored their experiences on a scale of 1�C5 (low to high, respectively).
The purpose of this was to evaluate any association between the reported strength of snus�� sensory effects and measured nicotine uptake. The questionnaire responses were obtained after 5, 10, 20, and 30 min of placing snus in the mouth. Blood Sampling Venous blood was collected by cannulation of the antecubital vein for both nicotine analysis (5 ml at each sampling time) and CYP2A6 genotyping (4 ml at visit 1, prior to any other blood sampling). Each sample was split into two aliquots. The sampling time points for nicotine analysis were preadministration (0) and at 5, 7, 10, 20, 30, 45, 60, 90, and 120 min after the start of each test product use. An additional sampling time point at 2 min was included while the cigarette was smoked.
Bioanalytical Methods Nicotine The bioanalysis of nicotine from plasma samples was conducted by Advanced Bioanalytical Service Laboratories Ltd., United Kingdom. Samples were analyzed by HPLC interfaced with an AB/MDS Sciex 4000 mass spectrometer. Quantification of nicotine was by peak area ratio. The determined lower limit of quantification for nicotine in plasma using this method was 0.5 ng/ml. Genotyping Genotyping of CYP2A6 alleles *1, *2, *4, *9B, and *12B was performed using whole blood samples collected into K2 EDTA VacutainerTM tubes, stored at ?20 oC and analyzed by QIAGEN Manchester Ltd., Manchester, United Kingdom (formerly DxS Ltd.). DNA preparation was performed using a QIAGEN spin column Dacomitinib kit. Prepared DNA samples were tested with a standard real time fluorescence assay to ensure quality of the test material. Specific DNA sequences were amplified by quantitative polymerase chain reaction (qPCR). All samples met the qPCR-QC acceptance criterion of Cycle Threshold <30. Pharmacokinetic and Statistical Analysis The pharmacokinetic analysis was conducted by Covance Clinical Research Unit (Leeds, United Kingdom) using WinNonlin Enterprise Version 5.2 (Pharsight Corporation, Mountain View, CA).