This article provides a quick guide to collect vapor emission for beginners. You will find information on how the specimens were prepared for research and what the results were.
Material and methods
Cambridge Filter Pads (CFP) exposure
Three test pieces were selected to study the impact of CFP: 1R6F reference cigarettes (University of Kentucky), Vype ePen3, and Vype eStick Maxx (Nicoventures Trading Ltd). A Borgwaldt LM1 Smoking Machine and an LM4E Smoking Machine (Borgwaldt KC, Hamburg, Germany) were used to collect the total particulate matter (TPM) from smoke and steam.
1R6F is an odorless blend cigarette with 0.721 mg nicotine per piece according to the International Organization for Standardization (ISO) and 1896 mg nicotine per cigarette according to the Health Canada Intense (HCI) smoking regimen. The Vype ePen3 is a closed modular e-cigarette that is activated by the push of a button and the Vype eStick Maxx is puff-activated. Both devices consist of two modules: a rechargeable battery and a cartridge with replaceable fluid. The Master Blend, containing 18 mg/ml nicotine, was in the Vype ePen3, and Toasted Tobacco, with 18 mg/ml nicotine, was used for the Vype eStick Maxx.
Sample preparation
The preparation of samples and analysis were carried out in accordance with the international standard No.7 – Determination of nicotine in mainstream cigarette smoke using CORESTA gas chromatographic analysis.
After collecting TPM, each CFP was cut into pieces and transferred to a 15 ml plastic tube containing 10 ml of extraction solvent (isopropanol, LC / MS grade, Carlo Erba) with N-decane (99% purity, Sigma-Aldrich.) (50 μg / ml). The tubes were vortexed for 30 min at 200 rpm. Thereafter, the devices under test were placed in an ultrasonic bath for 5 minutes. Then 1 ml of each sample was put through cellulose acetate filters (25 mm; 0.45 μm) and 100 μl of each extract was put to a vial with a conical insert for an autosampler.
Gas Chromatography with Flame Ionization Detection
This analysis was conducted using gas chromatography on a Shimadzu apparatus (model GC, 2010 AF) and a flame ionization detector. An Agilent J&W DB-HeavyWAX Intuvo GC column (30 mx 0.25 mm, 0.25 μm) was used.
Calibration curve
Nicotine solution with a concentration of 100 μg / μl was prepared by weighing 1 g of 99% pure nicotine (Sigma Aldrich) in a 10 ml volumetric flask and diluted to the required volume with acetone. The prepared solution was kept in the dark at a temperature from 0 ° C to 4 ° C. Solutions for the calibration of nicotine were the following concentrations of 0, 100, 200, 500, and 1000 μg / ml in 1 ml. The solutions consist of propane-2-ol with heptadecane of 99% purity (Sigma Aldrich, code 128503-100G). ) at a concentration of 50 μg / L.
Dosimetry effectiveness evaluation
To assess linearity, a calibration curve was constructed and the linear correlation coefficient (r2) was evaluated: the acceptance criterion was r2> 0.98. To assess the accuracy and precision, eight filters were added 100 µg of nicotine, and the other eight filters were added 500 µg each. For each nicotine level, accuracy was assessed based on the Relative Standard Deviation (RSD%) as a percentage between the standard deviation and the mean: the acceptance criterion was RSD% <10%. Accuracy was assessed on the basis of recovery (R%) as a percentage between the baseline value and the true value (100 μg or 500 μg): the acceptance criterion was 80% <R% <120%.
Nicotine normalization evaluation
Essential Component Analysis (PCA) was used to test the correlation of variables (nicotine on filters, TPM, difference in cartridge weight, or a number of puffs) and to determine if normalization of nicotine on filters for TPM was appropriate. One PCA was performed for electronic devices (Vype ePen and eStick) and one for 1R6F reference cigarettes. For the 1R6F, two exposure modes are included, ISO and HCI.
PCA component analysis was performed using RStudio software version 1.2.5033. PCA required the use of the regularized discriminant analysis (RDA) function. Before starting the analysis, the data were brought to a single standard. Filter samples were not scaled, and weighted variance scores were unchanged across all dimensions. The variables were scaled proportionally to their own values.
Comparison of different storage conditions
Statistical analysis was carried out using RStudio to assess the stability of nicotine under different storage conditions. Five groups of data were obtained, depending on the storage conditions for each test sample and the exposure mode. Three CFPs analyzed at time zero without any conditioning were included in control groups.
Four filters for each group were stored for 30 days under different conditions:
- at room temperature 25 ° C in a previously prepared solution (Group 1),
- at room temperature 25 ° C in a dry state (Group 2),
- at -20 ° C dry (Group 3) and (iv) at -80 ° C dry (Group 4)
CFPs from all four groups were tested for nicotine content. For each CFP sample, the amount of nicotine was normalized to the weight of TPM.
Test results
Dosimetry effectiveness
The test result showed that the linear correlation coefficient r2 of the calibration curve was 0.9999. The results showed the standard deviation (RSD%) and recovery (R%) if the nicotine level reaches 100 μg, 6.0%, and 94.6%, respectively, while the RSD% and R% at the 500 μg nicotine level are 4. 4%. and 109.7%.
Vapor impact in CFP
Before and after each e-cigarette aerosol exposure cycle, cartridge weights and CFP data were collected. To calculate the amount of TPM captured, the difference between the initial and final mass of CFPs that were exposed to steam was used. The results show that the mean ± standard deviation of TPM weights for the ePen and eStick is 82.7 ± 5.1 mg and 48.0 ± 7.4, respectively. And the mean values ± standard deviation of TPM weight, for 1R6F in accordance with the ISO and HCI modes, are 7.9 ± 0.4 mg and 30.8 ± 2.2 mg.
Nicotine normalization evaluation
PCA was analyzed by a linear transformation of four metrics, including nicotine on CFP, TPM, and cartridge weight difference or puffs, to simultaneously preserve maximum information from the individual variables. Major component 1 (PC1) and major component 2 (PC2), reflect the dynamics of changes, and the direction, not correlated with PC1, along which the samples show the greatest change.
The correlation diagram for e-cigarettes (PC1 73% and PC2 1.5%) showed that the amount of nicotine on the filters and TPM in the picture below has a high correlation. PCA correlates slightly with the difference in cartridge weight. Samples 1 to 19 refer to ePen and samples 20 to 38 refer to eStick. The two groups are divided into two different diagram areas.
Below you can see the correlation diagram for 1R6F: samples 1 to 19 refer to ISO exposure, and samples 20 to 38 refer to HCl.
The very correlation diagram for 1R6F (PC1 45% and PC2 9%) shows a high relationship between the amount of nicotine on the filters and TPM. PCA is only marginally dependent on the number of puffs.
Storing conditions analysis
The distribution of nicotine data normalized to TPM for Vype ePen was within the normal range in the following groups:
- 1 (p = 0.5719),
- 2 (p = 0.6298),
- 3 (p = 0.2742),
- 4 (p = 0.3741).
It turned out that the mean values of groups 2, 3 and 4 did not significantly differ from the mean values of the control group (group 2, p = 0.1319; group 3, p = 0.7277; group 4, p = 0.7408). The mean value of group 1 did not differ significantly from the value of the control group (p = 0.04016). Comparison of TPM normalized nicotine concentration on CFPs exposed to the Vype e-Pen using the Kruskal-Wallis test showed no significant difference between storage and control conditions.
Outline
As a result of this study, it was found that different effects and products influence the maintenance of the nicotine titer on CFP. However, decomposition of this substance can be avoided by refrigerating samples at -80 ° C for 30 days. This CFP storage method is recommended for protocol standardization and is necessary for interlaboratory studies of tobacco and nicotine-containing products. Also, during the tests, it turned out that the normalization of nicotine for TPM is a decisive factor for the correct assessment of the repeatability of the test. This made it possible to compare the amount of nicotine in the CFP with storage conditions and determine the key output values during vaping. Without the normalization of nicotine in the CFP, there would not be sufficient data to support storage testing.
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