E-cigarettes and pods
The European devices and differently flavored pods were purchased in local stores in Berlin and Sigmaringen, Germany, and online. The US-American variant was purchased in Tempe, Arizona.
Chemicals and standard substances
Used solvents or chemicals were of analytical or higher purity grade. 2-Propanol containing 0.3 g/L n-heptadecane, 2 g/L ethanol as internal standards and (S)-nicotine salicylate were purchased from LGC Standards (Teddington, UK), acetonitrile, sodium chloride, and orthophosphoric acid (85%) from Merck KGaA (Darmstadt, Germany). 2,4-Dinitrophenylhydrazine (moistened with 33% water) was bought from PanReac AppliChem (Darmstadt, Germany). Tris(hydroxymethyl)aminomethane, sulfuric acid (99.999%), (S)-nicotine, benzoic acid, benzoic acid-d5, and the 2,4-dinitrophenylhydrazone (DNPH) derivatives of acetaldehyde, acetone, acrolein, and formaldehyde were purchased from Sigma-Aldrich (Taufkirchen, Germany). Dimethyl sulfoxide was purchased from Honeywell Riedel-de-Haën (Seelze, Germany). Ultrapure water was prepared with a Milli-Q Integral Water Purification System (Merck KGaA, Darmstadt, Germany).
Aerosols were generated in two different laboratories, hereafter referred to as “lab A” (BfR, Berlin, Germany) and “lab B” (CVUA Sigmaringen, Sigmaringen, Germany). Both laboratories used a standard linear smoking machine that was designed for e-cigarettes (LM4E with PM1 piston pump, Borgwaldt, Hamburg, Germany). Experiments were performed according to CORESTA Reference Method 81 (CORESTA 2015) for the puffing regimen: 55 mL puff volume, 3 s puff duration, 30 s puff frequency, and rectangular puffing profile. E-cigarettes were placed in an angle of − 15° from a horizontal position. Except for carbonyl analysis, sessions of 20 puffs were taken with a clearing puff without e-cigarette at the end of each session. Between sessions, the liquid was allowed to cool down for approximately 10 min. The batteries were recharged after 8 and 6 sessions for the initial and the modified pods, respectively. Lab A compared two different custom adapters for a tight placement of the angular shaped e-cigarette mouth-piece on the filter holders as displayed in Fig. 5 in the Supplementary Material. One adapter was self-made with a heat shrinkable tubing (cross-linked polyolefin, HStronic GmbH, Schwäbisch Hall, Germany) that was prepared once and reused in combination with tape sealing (Parafilm, Bemis Company, Neenah, WI, USA). The second adapter was purchased from Borgwaldt (Hamburg, Germany) and used without additional tape sealing. Lab B used only the mouth-piece from Borgwaldt.
Determination of liquid consumption and total particulate matter (TPM), water and nicotine in the aerosol
TPM was collected on Ø 44 cm glass fiber filters (Borgwaldt, Hamburg, Germany). The filters in the filter holders and the e-cigarettes with pods were weighed before and after each session on analytical scales (LE225-0CE in lab A and CP225D-0CE in lab B, both Sartorius, Göttingen, Germany). TPM was calculated with the weight gain of the filters according to ISO 4387 (2019), consumption of the e-liquid with the weight loss of the liquid. Filters were extracted with 20 mL isopropanol containing internal standards (0.3 mg/mL n-heptadecane, 2 mg/mL ethanol) on a horizontal shaker (lab A: 3005, GFL, Burgwedel, Germany; lab B: SM-30 Control, Edmund Bühler, Hechingen, Germany) for 30 min at 80–100 rpm. The extracts were used for the determination of nicotine and water. Nicotine was quantified by gas chromatography with flame ionization detection (GC/FID). Lab A used a 6890 series from Agilent Technologies/Hewlett Packard (Agilent Technologies, Waldbronn, Germany) with a constant flow of 1.3 mL/min hydrogen (purity 99.999%, Linde, Pullach, Germany) on an HP-5 ms capillary column (30 m length, 250 µm inner diameter, 0.25 µm film thickness, 3 m pre-column, Agilent Technologies, Waldbronn, Germany). The temperature program started with 5 min at 100 °C, followed by a 30 °C/min ramp to 325 °C with 3.50 min hold. 1 µL filter extract was injected into a split/splitless injector at 250 °C and split ratio of 1:5 was used. FID was operated at 290 °C with a hydrogen flow of 30 mL/min, air flow of 300 mL/min, and a nitrogen (purity 99.999%, Linde, Pullach, Germany) make up flow of 20 mL/min. Lab B analyzed nicotine with flame ionization detection at 300 °C (7890A, Agilent Technologies, Waldbronn, Germany; 30 mL/min H2 flow, 99.999%; 400 mL/min air flow; 15 mL/min make up flow, N2, 99.999%; Air Liquide, Paris, France) and water with thermal conductivity detection at 250 °C (Agilent Technologies, Waldbronn, Germany; 15.5 mL/min reference flow, 5 mL/min combined flow) in one run after separation with a 7890A series gas chromatograph (Agilent Technologies, Waldbronn, Germany). 1 µL extract was injected into a split/splitless injector at 250 °C in splitless mode. Separation for nicotine analysis was performed by using an Rtx-VMS column (30 m × 0.530 mm, 3 µm film thickness, Restek GmbH, Bad Homburg, Germany), and for water an HP Plot Q column (30 m × 0.530 mm, 45 µm film thickness, Agilent Technologies, Waldbronn, Germany).The oven program started at 75 °C for 0.5 min, heated with a rate of 50 °C/min to 165 °C and held for 3 min, heated with 50 °C/min to 225 °C, held for 5 min, before it cooled down at 50 °C/min to 75 °C, followed by a 1 min hold. Flow rate of helium carrier gas (99.999%, Air Liquide, Paris, France) was 4.240 mL/min.
Determination of carbonyl compounds
Carbonyls were analyzed as described previously (Mallock et al. 2018) with liquid chromatography and UV detection on an RP-Amid column (Ascentis, 150 × 2 mm, 3 µm, Supelco, Bellefonte, PA, USA). Fractions of 40 puffs each were drawn through impingers that contained 35 mL of 2,4-dinitrophenylhydrazine (3.4 mg/mL in 45% acetonitrile with 0.35% orthophosphoric acid) each. After two clearing puffs, the content of both impingers was combined and incubated at room temperature for 30 min before reaction was stopped by addition of 2 mL tris(hydroxymethyl)aminomethane (tris) solution (16 mg/mL in 80% acetonitrile) to 8 mL of the sample. Calibration standards for carbonyl-DNPH-derivatives were diluted with the same DNPH/tris solution mixture to mimic effects of excess DNPH on the UV spectra.
Determination of benzoic acid in liquids and aerosol
Benzoic acid was quantified using headspace-solid phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS). 60 mg of sample liquid or self-prepared standard liquid (20 mg/mL nicotine in PG/VG 50:50 (w/w)) was weighed into 20 mL headspace vials and dissolved in 5 mL saturated sodium chloride solution containing 0.5 M sulfuric acid. 50 µL of isotope-labeled internal standard solution (8 mg/mL benzoic acid-d5) and/or calibration standard solution (4, 10, 15, 20 and 30 mg/mL benzoic acid) in DMSO were added and mixed. For analysis of benzoic acid in the aerosol, vaped filters were transferred into 20 mL headspace vials. For calibration, blank filters were used in combination with 40 mg or 80 mg standard liquid. Standard solutions were directly pipetted on the filter, followed by mixing with 5 mL saturated salt solution containing 0.5 M sulfuric acid. SPME was automated on an MPS2-XL autosampler (Gerstel, Mühlheim, Germany) with an incubation temperature of 80 °C and 1 min incubation time prior to 50 min headspace extraction by a polydimethylsiloxane/divinylbenzene fiber (Supelco, Bellafonte, PA, USA) with 250 rpm shaking only for the filter samples. The fiber was injected into a cooled injection system (CIS) 4 (Gerstel, Mühlheim, Germany) and desorbed for 5 min at 250 °C and a 1:50 split ratio. The GC 6890A (Agilent Technologies, Waldbronn, Germany) was equipped with a 30 m HP-FFAP capillary column with 250 µm inner diameter and 0.25 µm film thickness (Agilent Technologies, Waldbronn, Germany). After 5.5 min at 60 °C, the GC oven ramped with 15 °C/min to 240 °C and held for 15 min. The helium (purity 99.999%, Linde, Pullach, Germany) carrier gas flow was constant at 1 mL/min. The mass selective detector MSD 5975C (Agilent Technologies, Waldbronn, Germany) was equipped with an electron impact ion source (Agilent Technologies, Waldbronn, Germany) and operated with an ionization energy of 70 eV using a combined selected ion monitoring (SIM) and scan mode with a mass range from 29 to 300 m/z. Benzoic acid was quantified with the ion masses of 77 m/z and qualified with 105 m/z and 122 m/z. The internal standard benzoic acid-d5 was quantified with 82 m/z and qualified with 110 m/z and 127 m/z. Dwell time was 15 ms for each ion. Optimization of extraction parameters is summarized in the Supplementary Material.
Determination of density, pH value and nicotine content of the e-liquid
Liquids from the same batch were pooled for direct determination of density with an oscillating U-tube (DMA 500, Anton Paar, Graz, Austria). For quantification of the nicotine content in liquids, 300 mg liquid was diluted in 10 mL isopropanol with internal standards (0.3 mg/mL n-heptadecane, 2 mg/mL ethanol) and analyzed with the above mentioned GC/FID method in lab B. The pH value of a 1:20 dilution of liquids in ultrapure water was directly measured with a pH meter (765 Calimatic; Knick, Berlin, Germany).
Determination of propylene glycol and glycerol content of the e-liquid
E-liquids were analyzed by diluting a sample solution of approx. 5 mg/mL (precisely weighed) with methanol. The resulting solution was diluted by 1:1 with the internal standard solution containing 5 mg/mL 1,4-butanediol in methanol. 1 µL aliquot of this sample solution was injected into the split/splitless injector and analyzed by means of GC/FID. GC/FID analysis was performed on an Agilent 7890A gas chromatograph equipped with an FID detector and an autosampler (Agilent Technologies, Waldbronn, Germany). Separation was achieved on an HP-FFAP (25 m × 0.32 mm i.d. × 0.52 μm film) capillary column (Agilent Technologies, Waldbronn, Germany). GC/FID conditions were as follows: split mode, split ratio: 1:40; injector temperature: 230 °C; nitrogen (99.999%; Air Liquide, Paris, France) as carrier gas at a constant pressure of 0.7 bar. FID was operated at 250 °C (30 mL/min H2 flow, 99.999%; 400 mL/min air flow; 30 mL/min make up flow, N2, 99.999%; Air Liquide, Paris, France). The oven program started at 70 °C, held for 4 min. The temperature was raised by 10 °C/min up to 220 °C and held for 7 min, followed by a ramp of 30 °C/min to 70 °C. Total run time was 31 min.
Characterization of the pod construction
Resistance was measured between the connectors at the bottom of the pods with a 2010 DMM ohmmeter (PeakTech, Ahrensburg, Germany). For FT-IR analysis, the wick was removed from the pod, washed twice with ethanol, dried at 80 °C (FED 240, Binder, Tuttlingen, Germany), and analyzed with attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectroscopy using a Nicolet 6700 spectrometer (Thermo Electron Corporation, Madison, WI).