Generation of Substantially Metal-Reduced but Aldehyde-Rich Aerosol for E-Cigarette Toxicity Studies

Summary

Here, we establish that aerosolization of a commercial e-liquid by a pneumatic atomizer at 200 °C provides a metal-reduced aerosol that has aldehyde levels comparable to those found in real-world e-cigarette aerosol.

Abstract

When e-liquid is aerosolized in an e-cigarette for inhalation by the user, the aerosol becomes contaminated by a mixture of toxicants, including metals and aldehydes. A method to generate e-cigarette aerosol with high aldehyde but low metal content is lacking, yet is needed to untangle the potential independent effects versus toxicological interactions of aldehyde and metal exposure from e-cigarette aerosol mixtures.

The objective of this study was to establish a method for generating aldehyde-rich aerosols from e-liquids that lack, however, the large metal contamination typically found in e-cigarette aerosol. To aerosolize e-liquid at temperatures that promote aldehyde formation (up to 200 °C), a pneumatic atomizer was externally heated. Both a pure propylene glycol/vegetable glycerin (PG/VG) mixture and a commercial e-liquid were aerosolized. Aerosols were analyzed for known e-cigarette-associated toxicants, including 11 aldehydes using liquid chromatography-tandem mass spectrometry and 9 metals (Cr, Cu, Fe, Mn, Ni, Pb, Sr, V, Zn) using inductively-coupled plasma mass spectrometry.

Aldehyde levels in aerosols from the pneumatic atomizer only reached those in a real-world e-cigarette aerosol if the commercial e-liquid was aerosolized at 200 °C. Metal levels in the aerosols from the pneumatic atomizer were then at least 8 times lower than in e-cigarette aerosol from a "Mod" device operated at 50 W. Cu, Ni, and Pb levels were particularly low across all aerosol samples, with some levels below the method detection limit (MDL).

Aerosolization of a commercial e-liquid by a pneumatic atomizer at 200 °C produces aerosol that is low in metal levels (particularly Ni, Cu, Pb), but has aldehyde levels comparable to those found in a real-world e-cigarette aerosol.

Introduction

In the US, e-cigarettes are the most commonly used tobacco product among youth; in 2024, 5.9% of students reported using e-cigarettes¹. This is a public health concern because e-cigarette aerosols, which are inhaled by e-cigarette users, contain a broad range of toxic chemicals that, upon inhalation, can have various adverse health effects². Some toxic chemicals in aerosols may not only emanate from the original e-liquid formulation but also be introduced during device use. For instance, metals leach from the metallic heating coil used to aerosolize the e-liquid through the process of dissolution and evaporation into the aerosol³. In contrast, most of the aldehydes present in e-cigarette aerosol are formed from the base ingredients of e-liquids, propylene glycol (PG) and vegetable glycerin (VG), when the temperature of the e-liquid is increased to the boiling point of the e-liquid for the purpose of aerosolization⁴.

The presence of metals and aldehydes in e-cigarette aerosol is of particular concern because several of those are toxic, especially to the brain and other organs. For instance, the metals lead (Pb), manganese (Mn), copper (Cu), iron (Fe), nickel (Ni), and chromium (Cr) are known neurotoxicants which have been found to accumulate in the brains of mice exposed to e-cigarette aerosol, at levels previously described to cause neurotoxic effects⁵. High Mn and Pb exposure, Fe overloading in the brain, and Cu imbalance have all been linked to a higher risk for the development and/or progression of neurodegenerative diseases, e.g., Parkinson's disease (PD). This metal-induced neurotoxicity usually involves cellular mechanisms such as oxidative stress due to reactive oxygen species (ROS) overproduction, mitochondrial dysfunction, protein aggregation, and neuroinflammation^6,7,8.

The aldehydes acrolein and formaldehyde have also been linked to neurodegeneration^9,10,11, possibly leading to late-onset neurological diseases such as PD. As shown in a PD mouse model, aldehyde build-up can be toxic by promoting protein aggregation¹², and individuals with a mutation in the enzyme responsible for the conversion of acetaldehyde into acetic acid (ALDH2) may have a greater risk of Alzheimer's disease¹³. Even though it is known that metals and aldehydes can be neurotoxic individually, less is known about their effects as mixtures, and it appears quite challenging to untangle the independent and interaction effects of these two chemical groups in the context of e-cigarette aerosol exposure. To properly study chemical class-related toxicological effects, the capability is needed to generate simulated aerosols that are metal-reduced compared to aerosols from a chosen reference e-cigarette while matching its aldehyde levels.

The objective of this paper is to establish a method that, like in an e-cigarette, aerosolizes e-liquid at high temperature to generate an aldehyde-rich aerosol, which, however, lacks substantial metal contamination, to provide the toxicology field with a new tool to untangle the respective toxic effects of these chemicals within e-cigarette aerosol mixtures. To achieve this goal, we avoided aerosolizing the e-liquid by using a metallic heating coil, which would act as a source of metal contamination. Instead, we used a pneumatic atomizer¹⁴, which was heated externally to promote the formation of aldehydes from e-liquids. To evaluate our approach, we compared aldehyde and metal levels in aerosols from the atomizer to those from a "Mod" e-cigarette we used to expose rodents to e-cigarette aerosol.

Protocol

Setting up the pneumatic atomizer
All materials used in the study are listed in the Table of Materials file. To generate simulated e-cigarette aerosol without using a metallic heating coil that releases metals, we used a commercial pneumatic atomizer. To achieve the high temperatures needed to promote aldehyde formation, we heated the pneumatic atomizer as a whole by wrapping heating tape around it. To that end, we loosened the two screws, which secure the actual aerosol unit to its stand, such that the heating tape could be guided through the resultant gap instead of wrapping it around the stand. The temperature of the pneumatic atomizer was measured by attaching a platinum resistance temperature detector to its exterior surface, i.e., the temperature of the e-liquid was not directly measured. The thermal connection was improved by applying a small amount of thermal paste to the detector. The measured temperature was read in by a microcontroller board, which controlled a relay to supply the heating tape with 110 V line power if the actual temperature was smaller than a target temperature (between 50 °C and 200 °C). Figure 1 depicts the experimental setup.

Tubing materials that are needed to operate the pneumatic atomizer and that were originally supplied by manufacturer had to be replaced by more temperature resistant materials: 1) the liquid feed tubing (14-gauge PTFE) was replaced by high-temperature silicone tubing (1/8" OD), 2) the high-molecular weight polyethylene tubing sections (1/4" OD) of the excess liquid drain line and the filtered air supply were replaced by extreme-temperature hard plastic tubing (1/4" OD).

Figure 1: Diagram of experimental setup. Please click here to view a larger version of this figure.

Operating the pneumatic atomizer
Operating the pneumatic atomizer in the default recirculation mode could cause metal accumulation in the recirculating e-liquid, which is the excess, non-aerosolized e-liquid that flows through the excess liquid drain-off back into the e-liquid bottle. To prevent associated metal accumulation in the generated aerosol, the pneumatic atomizer was operated in non-recirculation mode, in which excess e-liquid flows into an added waste bottle. House compressed air was filtered and dried before supplying it to the atomizer via a filtered air supply, which consists of a coalescing pre-filter, a coalescing filter, a carbon filter, and a membrane dryer to remove solid particulate matter, oil and water droplets, and oil and water vapor. According to the manufacturer, the final carbon filtration removes 99.99995% of particles > 100 nm in size.

Aerosol collection
The pneumatic atomizer was used to generate aerosols from either a 70%vol PG/30%vol VG solution or a commercial, real-world e-liquid with 6% nicotine content that we used in previous research¹⁵. Aldehyde and metal levels of the aerosols produced by the pneumatic atomizer were compared to those in aerosols generated from the commercial e-liquid using a Mod e-cigarette, i.e., a modifiable device which can be refilled with e-liquid and the operating power of which can be adjusted. Recently, this Mod was used for chronic exposure of rodents to e-cigarette aerosol produced at 30 W⁵. In this study, the device was operated at a power output of either 30 W or 50 W.

In all the experiments, including those with the pneumatic atomizer and those with the Mod e-cigarette, aerosol samples were collected in triplicate using the method developed by Olmedo et al.¹⁶ for subsequent metal and aldehyde analyses. Aerosol samples were analyzed for 9 metals (Cr, Cu, Fe, Mn, Ni, Pb, Sr, V, Zn) using inductively-coupled plasma mass spectrometry (ICP-MS) and 11 aldehydes (acetaldehyde, acetone, acrolein, benzaldehyde, 2-butanone, butyraldehyde, crotonaldehyde, formaldehyde, hexaldehyde, propionaldehyde, pentanal) by liquid chromatography tandem mass spectrometry (LC-MS/MS) (see Supplementary File 1 and Supplementary Table 1 and Supplementary Table 2 for details). Potential metal transfer between the collected aerosol and the collecting tubing system was not a concern, except for Zn.

Electrochemical sensor for real-time aldehyde survey
A low-cost electrochemical sensor was used to compare in real time the aerosol aldehyde concentrations produced from different e-liquids at different temperatures, and thus be able to adjust more quickly the conditions that favor the generation of aldehydes as compared to offline analysis by LC-MS/MS. That sensor, according to the manufacturer, measures formaldehyde concentrations with a maximum reading of 5,000 ppb. The sensor could be directly connected to a computer via a USB cable, and formaldehyde concentrations could be viewed in real time using software provided by the sensor manufacturer. These real-time measurements enabled us to identify the optimal conditions for generating aldehydes with our pneumatic atomizer. To protect the sensor from the aerosol, the sensor was placed in a small box that allowed for the exchange of ambient air through a small-pore-size membrane that covered a hole in the box (while blocking aerosol droplets).

Statistical analysis
Box plots were used to visualize the distributions of the measured metal and aldehyde concentrations in aerosol and e-liquid (Figure 2). To get a sense of the overall aldehyde and metal concentration of an aerosol sample, we aggregated the data. We summed up the concentrations of the nine measured metals to determine a "total" metal concentration. Similarly, we summed up the concentrations of the eleven measured aldehydes to determine a "total" aldehyde concentration. All statistical analyses were performed using the R software version 4.3.3. Differences in metal concentrations across different aerosols and e-liquids were analyzed by t-test. When p-values were less than 0.05, differences were considered significantly different.

Results

Exploring the conditions that promote aldehyde formation
Aerosols were generated in four phases. At first, we used the pneumatic atomizer to generate aerosols from the PG/VG solution at 50 °C and 100 °C, and we used the "Mod" e-cigarette to generate aerosol from the commercial e-liquid at 30 W and 50 W operating power. As determined by LC-MS/MS, total aldehyde levels were much lower in the PG/VG aerosols than in real-world e-cigarette aerosol (Figure 2A).

Discussion

The total aldehyde concentration (11 aldehydes) of aerosols we generated with the pneumatic atomizer from commercial e-liquid at a temperature of 200 °C was on the order of those measured in aerosols from a Mod device operated at 30 or 50 W. In particular, acrolein occurred at the high levels observed in the real-world e-cigarette aerosols. However, levels of formaldehyde did not match those observed in the e-cigarette aerosols. Altogether, the levels of aldehydes reported in our study, which range from low ng/g to ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agilent 8900 ICPMS	Agilent	https://www.agilent.com/en/product/atomic-spectroscopy/inductively-coupled-plasma-mass-spectrometry-icp-ms/icp-ms-instruments/8900-triple-quadrupole-icp-ms?
Aldehyde/Ketone-DNPH standards	Supelco	ERA-028
Analyst software	Sciex	version 1.6.2
C18 column	Kinetex	https://www.phenomenex.com/part?partNo=00D-4475-AN	1.7 µm
Collision-type atomizer	TSI	Model 3076
Commercial e-liquid	Beyondvape, Baldwin Park, California, US	Pale Whale Vixen’s Kiss	purchased with a 6% nicotine content
Electrochemical sensor	Sensirion	SFA30
Extreme-temperature PTFE tubing	McMaster-Carr	5239K12	Extreme-temperature hard plastic tubing for chemicals, semi-clear Teflon® PTFE Plastic without Stripe, 3/16" ID, 1/4" OD,
Filtered air supply	TSI	Model 3074B
Heating tape	BriskHeat	TBIH051-020LD	Dual Element, Cloth Insulated Heat Tape, 2 inch x 1/2 inch, 156 Watts
High-temperature silicone tubing	Quickun	Pure Silicone Tubing, 1/16" ID, 1/8" OD
Mars 6	CEM	https://cem.com/mars-6
Microcontroller board	Arduino	Arduino Uno Rev3
Mod e-cigarette	Eleaf Electronics Co., Ltd., Shenzhen City, China	iStick Pico Plus, Melo 4S Tank, EC-A 0.3 Ω coil
Multiquant	Sciex	version 3.0.3
NexION 350S ICPMS	Perkin Elmer	NexION 350S
Nitric acid	Fisher Chemical	Optima	Ultra Trace Elemental Analysis grade
Platinum resistance temperature detector	Adafruit	PT1000
Qtrao 6500 LC-MS/MS System	Sciex	https://sciex.com/products/mass-spectrometers/qtrap-systems/qtrap-6500-system	Tandem mass spectrometer
Shimadzu LC	Shimadzu	https://www.shimadzu.com/an/products/liquid-chromatography/index.html