A Method for Measuring Nicotine in Animals Following a Vaping Model

In recent years, the use of electronic cigarettes has rapidly increased, both as an alternative to and a means of quitting tobacco smoking. Thus, research is necessary to examine the consequences of nicotine vaping. Previous preclinical research models of nicotine exposure largely utilized injected nicotine; however, recent advances in the development of rodent vaping chambers have enabled nicotine studies to more accurately mimic typical human nicotine use. However, in order to understand the clinical significance of this work, these studies need to be accompanied by pharmacokinetic studies that examine concentrations of nicotine that are reached in the plasma as well as the brain. Therefore, we developed a method by which nicotine delivered to mice through One E-VapeTM delivery system could be quantified in these matrices. Briefly, nicotine freebase was dissolved in e-liquid containing a 50/50 blend of propylene glycol and vegetable glycerin at a concentration of 6 mg/mL. The mice received the vapor in their home cage, which was placed directly into the vapor chamber. Six puffs total were delivered over ten minutes, after which the mice were removed once the last puff completely cleared the chamber. Mice were sacrificed via cervical dislocation directly following removal from the vapor chamber. Plasma and brain samples were collected and stored at -80oC until analysis. Briefly, nicotine was extracted from plasma and brain homogenate via solid phase extraction using Clean Screen DAU extraction columns. Chromatographic separation utilized an Atlantic HILIC Silica column (3 micron, 2.1 x 100mm) with an isocratic elution of acetonitrile (86%) and 5 mM ammonium formate, pH 3 (14%) at a flow rate of 0.5 mL/min. Mass spectrometric detection was achieved using a Shimadzu IT-TOF system with the electrospray source running in positive mode. Data acquisition utilized a direct MS-MS method using m/z 163.1017 as a precursor ion for nicotine, and m/z 167.1401 for the internal standard, d4-nicotine. The calibration curve ranged from 100 ng/ml to 5 ng/ml. These conditions allowed for a lower limit of detection (LLOD) less than or equal to 1 ng/mL and a lower limit of quantification (LLOQ) of less than or equal to 10 ng/mL. Moreover, the method was successfully used to detect nicotine in both plasma and brain tissue delivered through the One E-Vape vaping system. Thus, it will be useful in further studies examining the effects of nicotine vaping in rodent models