Sampling and chemical analysis
A total of 54 e-cigarette liquid samples was analysed. This includes e-cigarette liquids (n = 20) submitted to the CVUA Karlsruhe for official medicines and tobacco control purposes in Baden-Württemberg, Germany. Furthermore, an internet-based market research was conducted to identify products offered to German consumers. To do this, we searched for the German terms “E-Zigaretten Liquids kaufen”, “E-Liquids Kaufen”, “E-Liquids Flash”, “E-Liquids Hanf”, “E-Liquids Marihuana” using the web platforms google.de, amazon.de and ebay.de. We also included search terms such as “E-Liquids Cialis”, “E-Liquids Vitamin“, because the German Federal Institute for Risk Assessment (BfR) recently found pharmacological active substances such as tadalafil, caffeine or vitamins in these products [
23]. In total, 13 on-line shops were selected, that are selling such liquids. Most of the shops (n = 8) were located in Germany, one was located in Spain, three were located in the United Kingdom and one was located in Romania. Based on the invoices, it seems that several shops, though being located in countries outside Germany, had local partners, who handled the shipping and returning of the good, if necessary. The liquids were sent to us in envelopes or parcels. Most of the products were labelled as “Liquids for E-Cigarettes” or simply “E-Liquid”, but in some cases the products did not contain any labelling and in one case the products were labelled as “Air freshener”.
From the identified on-line shops, we selected samples using a risk-oriented approach. For example, we preferentially obtained samples where the presence of bioactive flavour compounds could be assumed. Additionally, several samples suspected of containing illegal or unusual substances were bought, such as products labelled as “Marihuana-Flavour”, “Mary Jane Flavour”, “Vitamin-Mix” or “Multi Vitamin”. Furthermore, several samples with tobacco and beverage flavour (such as cola, wine, energy drink or absinthe flavour) were included. We tried to obtain all varieties of e-cigarette liquids regarding declared nicotine content. We obtained several samples, claimed as nicotine-free and samples with medium or high nicotine content (the labelled nicotine content varied between 6 and 54 mg/ml).
NMR analysis of electronic cigarette liquids
The chemical analysis was conducted using NMR spectroscopy based on a procedure previously developed for analysis of alcoholic beverages [
24].
All solvents and reagents used were in pro analysis quality: nicotine, menthol, safrole (Sigma Aldrich, Steinheim, Germany), propylene glycol, 1,3-butanediol, 1,3-propanediol, ethylene glycol, glycerol, ethyl vanillin, camphor, α-thujone, coumarin, diethyleneglycol (Fluka, Buchs, Switzerland).
To obtain full quantitative information about e-cigarette liquids composition, we decided to apply two separate sample preparations for each sample. To measure water-soluble compounds (including nicotine), 60 μL of a sample was mixed with 480 μL of distilled water and 60 μL of NMR buffer (pH 7.4; 1.5 M KH2PO4 in D2O, 0.1 % 3-(trimethylsilyl)-propionate acid-d4 (TSP), 3 mM NaN3). Next, to get an overview of lipophilic substances, the following sample preparation was used: 100 μL of a sample was mixed with 800 μL CDCl3 containing 0.1% tetramethylsilane (TMS). After filtration (when necessary), 600 μL of the solution was poured into an NMR tube for direct measurement in both cases.
Stock standard solutions were prepared at a final concentration of about 10,000 mg/L in distilled water (nicotine bitartrate, propylene glycol, 1,3-butanediol, 1,3-propanediol, ethylene glycol, diethylene glycol and glycerol) or in deuterated chloroform (menthol, ethyl vanillin, coumarin, camphor, safrole and α-thujone). Calibration solutions were prepared by diluting the standard solutions in water or in deuterated chloroform and were measured as the authentic samples. The calibration curves were evaluated by integrating specific resonances of the selected compounds against TSP (in water) or TMS (in CDCl3) as an intensity reference.
All NMR measurements were performed on a Bruker Avance 400 Ultrashield spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5-mm SEI probe with Z-gradient coils, using a Bruker Automatic Sample Changer (B-ACS 120). All spectra were acquired at 300.0 K.
NMR spectra of the aqueous solutions were acquired using Bruker standard water suppression 1D noesygppr1d pulse sequence with 64 scans (NS) and 4 prior dummy scans (DS). The sweep width (SW) was 19.9914 ppm and the time domain of the FID was 65536 (65 k). For the spectra acquisition of chloroform-dissolved samples, the Bruker experiment zg30 was used. After application of 2 dummy scans (DS), 8 free induction decays (FIDs) (NS = 128) were collected into a time domain of 131072 (131 k) complex data points using a 20.5503 ppm spectral width (SW) and a receiver gain (RG) of 101.
The data were acquired automatically under the control of ICON-NMR (Bruker Biospin, Rheinstetten, Germany), requiring about 40 min per sample for two measurements. All NMR spectra were phased and baseline-corrected.
The spectra of samples were compared to the spectra of the standards. Separated peaks corresponding to each substance were identified and integrated against TSP/TMS as an intensity reference using TopSpin v. 3.1 (Bruker Biospin, Rheinstetten, Germany). NMR ranges used for identification/integration for all compounds are listed in Table
1. Furthermore, all samples were screened for a range of small molecular weight molecules such as formaldehyde, acetaldehyde and acrolein.
Table 1
Selected resonances, limits of detection (LOD) and limits of quantification (LOQ) for compounds in e-cigarette liquids
1 | Nicotine | Water | 8.68-8.60 (multiplet) | 1.6 | 5.5 |
2 | Propylene glycol (1,2-propanediol) | 3.47-3.42 (multiplet) | 2.1 | 6.9 |
3 | 1,3-Butanediol | 1.75-1.70 (multiplet) | 2.3 | 7.6 |
4 | 1,3-Propanediol | 1.85-1.75 (multiplet) | 0.96 | 3.2 |
5 | Ethylene glycol | 3.69-3.67 (singlet) | 0.17 | 0.56 |
6 | Diethylene glycol | 3.78-3.73 (multiplet) | 0.51 | 1.7 |
7 | Glycerol | 3.82-3.75 (multiplet) | 2.6 | 8.7 |
8 | Menthol | Chloroform | 2.00-1.92 (multiplet) | 12 | 40 |
9 | Ethyl vanillin | 9.81-9.83 (singlet) | 1.0 | 3.4 |
10 | Coumarin | 7.80-7.65 (multiplet) | 3.2 | 10 |
11 | Camphor | 2.40-2.30 (multiplet) | 13 | 44 |
12 | Safrole | 6.80-6.60 (multiplet) | 2.6 | 8.6 |
13 | Thujone (sum of α- and β-diastereomers) | 2.12-2.09 (singlet) | 3.4 | 11 |
We conducted a detailed validation for the determination of nicotine in e-cigarette liquids. For this substance the limits of detection (LOD) and quantification (LOQ) were calculated from the residual standard deviation of the regression line [
25]. To assess reproducibility, standard solutions as well as an e-cigarette liquid sample were analyzed several times daily (
n = 5). The recovery rate was ascertained by adding nicotine standard solution at four different concentrations to a real sample.
For other substances, the LOD and LOQ values were determined as signals for which the signal-to-noise ratios (SNR) are 3 and 10, correspondingly. SNR were calculated using the Bruker sino routine implemented in the Topspin 3.1 software package (Bruker Biospin, Rheinstetten, Germany). The limits of the noise and signal regions were located near to each other and were set manually for each spectrum.
The linearity of the calibration curves was evaluated in the range covering the concentrations found in the investigated products.
Risk assessment
The methodology for comparative quantitative risk assessment was based on a previous study conducted for compounds in alcoholic beverages [
26] with the exception that probabilistic exposure estimation was conducted [
27]-[
29].
The toxicological thresholds, preferably benchmark doses (BMD) or if unavailable no observed effect levels (NOEL), no observed adverse effect levels (NOAEL) or lowest observed adverse effect levels (LOAEL), for the selected substances were typically identified in monographs of national and international risk assessment bodies such as EFSA, OECD SIDS, JECFA, and ATSDR [
30]-[
35], and if unavailable – as in the case of thujone - taken from an own study [
36].
The MOE approach was used for risk assessment [
37],[
38]. The MOE is defined as the ratio between the lower one-sided confidence limit of the BMD (BMDL) or NOEL/NOAEL/LOAEL and estimated human intake of the same compound.
The exposure was estimated for daily users of e-cigarettes based on the contents found in our chemical analysis. Similar to the approach of Medeiros Vinci et al. [
39] for probabilistic human exposure assessment of food contaminants, best fit distributions were applied to the substance contents and the resulting risk functions were entered into the probabilistic analysis. Further assumptions were literature data about typical e-cigarette liquid use per day [
40] and literature data about vaporization percentage [
19]. The bodyweight was assessed as normal distribution with average of 73.9 kg and standard deviation of 12 kg for males and females [
41]. All risk functions were truncated at zero because negative values are factually impossible. Monte Carlo simulations were performed with 10,000 iterations using Latin Hypercube sampling and Mersenne Twister random number generator. Calculations were performed using the software package @Risk for Excel Version 5.5.0 (Palisade Corporation, Ithaca, NY, USA).