Nicotine delivery and relief of craving after consumption of European JUUL e-cigarettes prior and after pod modification

Aim and ethics

Aim of the study was to get information about the addictive potential and addiction satisfaction of the European JUUL (“initial” and “modified” versions) compared to a tobacco cigarette. Therefore, we analyzed nicotine delivery of these products, especially in the acute phase, by examining venous blood plasma. The study was approved by the ethics committee of the LMU Munich (Amendment to project number 72-15) and performed in accordance with the principles of the Declaration of Helsinki in the currently valid version. It was registered at the DRKS (DRKS00017432). Informed consent was obtained from all participants before participation in the study.

Study products and groups

The study was designed as a single-center three-arm study. The products we used were (a) commercial, combustible tobacco cigarettes (Marlboro Red, Philip & Morris), (b) JUUL e-cigarettes with the new technology (JUUL “modified”) with rich tobacco flavor, and (c) JUUL e-cigarettes with the old technology (JUUL “initial”) with rich tobacco flavor. As previously published, the wick used in “modified” JUUL pods consist of a different material than the wick used in the “initial” JUUL pods32. Participants received the same instruction for use of both JUUL variants according to the producer manual. Products were purchased in local stores in Berlin and Munich, Germany, and online.


This single-center three-arm study included 15 active smokers and 17 active e-cigarette users who were tested with one or both products. 15 sessions were performed for cigarettes, 15 for the modified JUUL, and 11 for the initial JUUL version. This gives a total of 41 experimental sessions. Data from one participant that did not show any increase in nicotine plasma concentration were excluded from analysis. The participants were divided into either the tobacco cigarette group or one of the e-cigarette groups according to the product they normally used. The participants were recruited for participation in the study via advertisement with flyers and the internet. Participants were enrolled in the study after inclusion and exclusion criteria had been checked and participants had provided written informed consent. Inclusion criteria for all volunteers: Age between 18 and 55 years, 12 h of abstinence (e-cigarette and tobacco cigarette consumption), CO levels < 5 ppm (measurement in the expiratory air using a micro-smokerlyzer; Bedfont Scientific Ltd., Anif, Austria) to verify smoking abstinence, and ability to give consent. Special inclusion criteria for electronic cigarette users were e-cigarette use for > 3 months, daily consumption, no daily consumption of conventional tobacco cigarettes for > 3 months. Special inclusion criteria for tobacco cigarette smokers were daily smoking for > 5 years, consumption of > 10 cigarettes/day.

Exclusion criteria for both (electronic cigarette users and tobacco cigarette smokers): Participants under 18 or over 55 years of age, acute psychiatric illness according to ICD-10/DSM IV, other serious psychiatric disorders, acute suicidality, existing pregnancy, breastfeeding, drug, medication, or alcohol abuse at the time of the study, malignant cancer in the past 5 years, serious internal illness, especially cardiovascular diseases, such as manifest arterial hypertension, severe heart disease (DCM, history of heart attack), pacemaker implantation, respiratory failure, severe active infectious disease. E-cigarette users were invited to participate in both JUUL study arms. Thus, 9 participants used both JUUL variants, 6 participants used only the “modified” JUUL, and 2 participants used only the “initial” JUUL.

Study design and questionnaires

Clinical data were collected during January and September 2020. Two appointments were scheduled for the test subjects to participate in the study. The first appointment was considered a screening, whereas the actual measurement took place at the second appointment. Usual smoking or e-cigarette consumption behavior was enquired with standardized and specially designed questionnaires.

An initial questionnaire at the screening appointment served on the one hand to assess sociodemographic data such as sex, age, weight and known pre-existing illnesses and on the other hand to assess smoking and e-cigarette consumption behavior. For example, the frequency of smoking or vaping and preferred manufacturers were assessed. At the screening appointment, physical dependence on nicotine was assessed with the Fagerström Test of Nicotine Dependence (FTND) according to Heatherton et al.34. No validated version of the FTND exists for e-cigarette consumption. Thus, instead of the FTND, an adapted but unvalidated questionnaire for e-cigarettes was used for participants in the JUUL study arms. The Questionnaire on Smoking Urges (QSU-G) in the German version by Müller et al. was used to assess craving35. QSU-G was assessed before and immediately after the vaping/smoking sessions. Further details on QSU-G and its evaluation are given in the Supplementary Information. Immediately after vaping, participants rated negative effects (side effects) of the e-cigarette on a visual analog scale (VAS) ranging from 0 (no effect) to 10 (strongest effect). The VAS used in this study was based on the one used in previous studies on e-cigarettes36,37 and enquired urge to vomit, nausea, perspiration, headaches, palpitations, cold hands or feet, salivation, dizziness, irritation of the throat or mouth, and lightheadedness.

E-cigarettes were weighed (MC 1, Sartorius, Göttingen, Germany) before and after the measurement to determine the liquid consumption. Nicotine doses were calculated by considering liquid density (1.16 g/cm3) and the measured nicotine concentration in the respective e-liquid (17.69 mg/mL for “modified” JUUL and 17.20 mg/mL for “initial” JUUL)32.

Puff topography

The consumption sessions were carried out according to Fig. 1. A puff duration of 3 s was selected according to a recent study on pod e-cigarettes38 and to a well-established regime for machine puffing of e-cigarettes39. It was ensured that the puffs were 3 s long and that the blood samples were taken after the completed puff. In total, 10 puffs were taken, heart rate and blood pressure were measured 4 times, and 9 blood samples were taken. A metronome was used to standardize the duration of the inhalations by providing an acoustic signal at the beginning and end of inhalation. The study investigator instructed all study participants to inhale in exactly the same way at each inhalation and study visit. Participants were instructed to inhale the product aerosols into their lungs.

Figure 1

Blood sampling

A peripheral venous cannula was inserted to allow blood samples to be taken at short intervals. To determine nicotine, cotinine, and trans-3′-hydroxycotinine (hydroxycotinine) levels, a total of nine blood samples with 7.5 mL each were taken at various time points before, during and after smoking/vaping as presented in Fig. 1. They were carried out in accordance with the generally applicable hygienic standards using Safety Multifly cannulas and S-Monovettes. Blood was placed on ice immediately after sampling until centrifugation (10 min, 1500g, 4 °C). Internal standard mix (10 µL of 500 ng/mL nicotine-d3, cotinine-d3, hydroxycotinine-d3 in acetonitrile) was added to plasma samples (990 µL).

Analysis of nicotine, cotinine, and hydroxycotinine plasma concentrations

Nicotine and its main metabolites cotinine and hydroxycotinine were analyzed using LC–MS/MS with a validated method as published previously40. Blood samples were centrifuged at 1500g and 4 °C for 10 min to obtain plasma. 990 µL plasma was spiked with 10 µL internal standard mix (500 ng/mL (±)-nicotine-(methyl-d3), (±)-cotinine-(methyl-d3), and trans-3′-hydroxycotinine-d3 in acetonitrile) at LMU in Munich and shipped on dry ice to the BfR in Berlin. For protein precipitation, 100 µL ice-cold methanol were added to 50 µL plasma sample and then centrifuged at 4 °C and 14,000g for 15 min (Centrifuge 5427 R, Eppendorf, Wesseling-Berzdorf, Germany). Supernatant was diluted 1:1 with mobile phase A (see below) prior to injection of 25 µL into the LC–MS/MS system (LC: Prominence series from Shimadzu, Kyoto, Japan; MS/MS-System: API4000QTrap, AB Sciex, Framingham, MA, USA). For separation, a Luna Phenyl-Hexyl Column (150 mm length × 4.60 mm I.D., 3 µm particle size, 100 Å pore size; Phenomenex, Torrance, CA, USA) with an according guard column (Phenomenex, Torrance, CA, USA) was used at 45 °C at a flow rate of 1 mL/min. As eluent A, 5 mM ammonium acetate in water, pH adjusted to 4.50 ± 0.02 with formic acid was used and for eluent B methanol. The gradient was as followed: Started at 10% B, increase for 1 min to 30% B, hold for 1 min, increase for 2 min to 95% B, hold for 2 min, decrease for 0.2 min to 10% for 0.2 min, and a hold for 2.8 min. ESI–MS/MS parameters are provided in the Supplementary Information. Nicotine, cotinine, and hydroxycotinine were quantified using a matrix matched calibration.

Machine vaping

To mimic vaping, 10 puffs were taken from JUUL devices equipped with 5 freshly opened “modified” JUUL pods or 4 freshly opened “initial” JUUL pods using a linear vaping machine for e-cigarettes (LM4E with PM1 piston pump, Borgwaldt, Hamburg, Germany). By the time the machine vaping part was performed, “initial” pods were already taken off the market. Thus, only 4 pods could be analyzed. CORESTA Reference Method 81 was applied: puff duration 3 s, puff frequency 30 s, puff volume 55 mL, rectangular puff profile39. Emissions were collected on glass fiber filter pads (Borgwaldt, Hamburg, Germany) that were exchanged after 2 puffs during the 30 s inter-puff interval. Total particulate matter (TPM), the weight gain in the filter, was determined by weighing (LE225-0CE, Sartorius, Göttingen, Germany). Nicotine dose was calculated by dividing by liquid density (1.16 g/cm3) and multiplication with the nicotine concentration in e-liquid (17.20 mg/mL for the initial and 17.69 mg/mL for the modified version) as previously determined32. As previously shown, the nicotine content in the aerosol can be calculated on the basis of the liquid consumption leading to similar results as determined with GC-FID32. This is in line with other studies41. Further, we have previously shown that weight loss of the liquid (liquid consumption) and weight gain of the glass fiber filter (TPM) are comparable32, also in line with the literature41.

Pharmacokinetic (PK) parameters and statistical analysis

Statistical analysis was performed with Statistical Software Program System (SPSS) version 21.0. Data derived from QSU-G were analyzed with the t-test for paired samples. Areas under the plasma concentration–time curve (AUC) were calculated after baseline correction (subtraction of Ct0) applying the linear trapezoid rule. Cmax and tmax were the highest nicotine concentrations per individual plasma curve and the according time points. Participants were asked to stay nicotine abstinent overnight. However, nicotine PK parameters were reported with and without baseline correction (subtraction of Ct0) to control potential high nicotine baseline effects, possibly due to intensive smoking on the previous evening. For statistical analysis of Cmax and AUC, geometric mean and CV were used, and a two-sided unpaired t-test was used with lognormal values to test for statistical significance. For mean plasma curves, arithmetic means of baseline corrected concentrations at each time point were calculated. Nicotine metabolic ratio (NMR) was calculated as a surrogate for CYP 2A6 metabolic activity42,43 by dividing hydroxycotinine plasma concentration by cotinine plasma concentration at t0 when metabolites were detected. For NMR and other participant characteristics, median and IQR were calculated. Arithmetic mean and standard deviation were used for liquid consumption, nicotine dose, and TPM.

Source link

Leave a Reply

Your email address will not be published. Required fields are marked *