New Colorado Regulations for the Measurement of Heavy Metals in Cannabis Vaping Aerosols, A Realistic and Practical Assessment: Part 2

Part 1 of this series on the new Colorado regulations for heavy metals in cannabis vaping aerosols examined the fundamentals of vaping and the process of converting a vape liquid into an aerosol and the difficulties associated with characterizing the metal content. Part 2 will focus on the challenges of trapping and collecting the aerosol without contaminating the sample, and how best to validate the process using standard methods developed by the tobacco industry for electronic nicotine delivery systems (ENDS).

E-Cigarette aerosol generation and collection

If vaping aerosol analyses are to provide robust data, the methodologies and instrumentation must be scientifically sound and defensible. While no vaping machine puffing regimen mimics a human vaper, it is imperative that the data collected follows a standard and reproducible procedure. There is an ISO standard - Method, 20768:2018, which was specifically developed for the routine testing of vapor products with an analytical vaping machine ¹. However, there are no cited references in the public domain to understand how it is being applied to this type of analysis. So another standard method is CORESTA Method 81, which defines the requirements for the generation and collection of e-cigarette aerosol for analytical testing purposes ². It is similar in functionality to the ISO Method and has been widely used with acceptable specifications, including prescribed limits for pressure drops, puff volume, puff time, and puff profile for generation and collection of reproducible ENDS aerosol results. In addition, there are many citations in the open literature on the use of CORESTA Method 81 for carrying out investigations of metal particulates in END systems, many of which are referenced in this article. The testing protocol described in the CORESTA method requires a 3 second; 55 mL puff every 30 seconds with a machine pressure drop no greater than 300 Pascal (Pa) and a pressure drop across the aerosol trapping assembly no greater than 900 Pa with a rectangular puff profile at a linear airflow of 140 mm/s. Since the heating elements of some ENDS and ECDS devices are activated by sufficient airflow, the heating elements in these devices will not generate aerosol until the airflow reaches the rate that triggers the heating element. Therefore, a rectangular puff profile is required and should be verified with a 1000 Pa restrictor in place. The sum of the times required for airflow through the device to ascend to 18.5 mL/s and to descend to baseline may not exceed 10% of the puff. This assures that air flow-activated heating elements begin heating at the beginning of the puff. It should also be emphasized that since CORESTA Method 81 was written assuming the use of standard glass fiber filters for trapping organic constituents of aerosols, they should be replaced with traps that are appropriate for inorganic aerosol constituents.

The major specifications described in CORESTA Method No 81 are shown in Table 1.

Table 1: Major specifications defined in CORESTA Method No 81 ².

Puff Specification	Value
Puff Profile	Rectangular with a pressure drop device of 1000 Pa ± 50 Pa.
Profile Maximum Flow Rate	18.5 ml/s ± 1 ml/s
Puff Duration	3 s ± 0.1s
Puff Volume	55 ml ± 0.3 ml
Puff Frequency	1 puff every 30s
Puff Number	Total number of puffs collected from an e-cigarette

There are many different e-cigarette aerosol vaping systems on the market, which can accommodate multiple e-cigarettes or vaping devices and are suitable for ECDS. An example of one is shown in Figure 2 which is the Cerulean CETI 8 - an 8-port aerosol testing machine ³.

The weight of each element in nanograms (ng) in the collected aerosol is then calculated based on the number of puffs. For example, a common unit to compare different ENDS or ECDS is either ng/10 puffs or ng/50 puffs, depending on the purpose of the analysis.

Collecting and trapping hydrophobic liquids from ECDS 

As mentioned previously, analyzing E-liquids from ENDS and ECDS is a fairly straight forward dilute and shoot method using a nitric acid/hydrochloric acid mixture, because diluents like propylene glycol and vegetable glycerin are water-soluble. Even liquids from ECDS are not over complicated because the hydrophobic cannabinoid oils and diluents like MCT oil can either be diluted with a suitable organic solvent (as long as the sample introduction system is optimized) or the oils can be digested in a microwave digestion oven. Both these approaches are more time consuming but definitely achievable with an experienced analyst. However, to sample a hydrophobic aerosol of different oil mixtures is an order of magnitude more difficult. Filters tend to become clogged by viscous condensates. There is a good chance that the resulting collected material will be oily in nature which might require an organic solvent to make sure that will also maintain the metallic contaminants in solution. 

This is exemplified by the trapping methodology described in a recent publication by Pappas and coworkers, which is based on a modification of the method used for ENDS ^{4, 5}. The trap consists of a 518 cm length of 3.97 mm i.d. FEP tubing that is connected to the vaping machine syringe pump. This tubing is thoroughly rinsed with 2% v/v nitric acid + 1% v/v hydrochloric acid before and between each use. The vaping devices are connected to the FEP tubing from the vaping machine with Tygon tubing that has been thoroughly soaked in 2% v/v nitric acid + 1% v/v hydrochloric acid, since untreated Tygon has leachable metals. The FEP tubing traps aerosol and particles by condensation. The trapped aerosol is rinsed from the tubing with 3 x 8 mL rinses from a PFA (Perfluoroalkoxy) syringe with 2% v/v nitric acid + 1% v/v hydrochloric acid into a class A 25 mL polymethylpentene (PMP) volumetric flask, and dilution to 25 mL with the same acid solution. The modification of this method for oily ECD aerosols includes an initial tubing rinse with 5 mL diethylene glycol monoethyl ether (DEGMEE). This solvent has low volatility, is an excellent solvent for oils, and is water-miscible. Unfortunately, no high purity grade with respect to trace metals is commercially available, so it must be distilled from a high purity fused silica quartz distillation flask prior to use, which would present difficulty with regard to regulatory analyses. DEGMEE dissolves and dilutes the oil droplets from up to 50 CORESTA Method 81 vaping puffs as it passes through the condensation tube into a 50 mL PMP volumetric flask. The remnant in the condensation tube is then followed by 4 x 8 mL rinses with 2% v/v nitric acid + 1% v/v hydrochloric acid into the same 50 mL flask, and dilution to 50 mL with the same acid solution. Since DEGMEE is both oil and water-miscible, it solubilizes or at least emulsifies the oil with the aqueous acid for analysis. Calibration standards are prepared in 2% v/v nitric acid, + 1% v/v hydrochloric acid + 10% DEGMEE for the purpose of matrix matching with the same solution obtained after rinsing aerosol metals from the condensation tube.

Note: The data from this study used ng/50 puffs for comparison purposes.

What analytes are considered important to monitor?

As mentioned previously, metal components of ENDS and ECDS which could be exposed to the liquid inside the device cartridge or pod include stainless steel (mostly iron, chromium, and nickel), nichrome (nickel and chromium), kanthal (iron, chromium, and aluminum), brass (copper and zinc), and solder (tin and lead). Occasionally, wires are coated with silver and in some cases, silica-based ceramic heating elements are used as well as gold alloy coatings on electrical contacts.

Currently the regulated heavy metals for cannabis and cannabinoid products in most US states include lead, arsenic, cadmium and mercury, while Maryland adds chromium, selenium, barium and silver and New York adds zinc, antimony, copper, chromium and nickel ⁶. So the question to ask is, what should be included in a panel of elements for ECDS aerosols? Clearly, the point of testing vaping devices is to find out what toxic substances are being corroded and transported to the user? However, can it be assumed that the vaping liquid (cannabinoid plus diluent oils) has been tested for at least the big four (lead, cadmium, arsenic and mercury) as part of the regulatory process in that state?  I’m not sure this is always the case considering the many product recalls for CBD oils over the past 6-12 months ⁷. But assuming they have been tested for the state required panel and are below the maximum allowable limits, if any of these elements show up in the device aerosol, they will most probably have come from the vaping process. However, besides lead, which is a common constituent of solder, it is highly unlikely that components inside the device will contain elements like cadmium, arsenic or mercury, unless there has been some kind of contamination involved with sampling and/or measurement process. Of course, that could happen, particularly if the optimum trapping and collection technology is not being used. So it would be legitimate to include all the likely metal candidates to the regulated list for the state, based on the design of the vaping device, but it is not clear what limits should be set for those additional elements. You could select, the United States Pharmacopeia/International Conference on Harmonization (USP/ICH) permitted daily exposure(PDE) limits for inhaled pharmaceuticals ^{8, 9}, but they would be meaningless when current research on nicotine devices has shown that these metals are predominantly in the form of metal and metal oxide nanoparticles. Pappas and co-workers did a very thorough evaluation and confirmed this using scanning electron microscopy fitted with an energy dispersive X-ray spectrometer, together with dynamic light scattering and single particle ICP-MS studies ¹⁰. 

Additionally, with an e-cigarette aerosol testing machine, how would the weight of metal per number of inhaled puffs, be compared with a traditional regulated maximum allowable limit? So for that reason, there would probably need to be an additional set of regulations just for ECDS, based on the nanograms per number of puffs.  And one final point to emphasize, which is an ongoing problem in the field of cannabis testing…What measure of validation protocols would be appropriate? We know that it is not meaningful to spike the vape liquid and expect good recoveries, because the elements are not volatilized and transported consistently and efficiently to the trapping and collection device. So, at this point, it is not clear what could be used as a set of validation procedures for characterizing vaping aerosols for heavy metals.

The final part of the series will look at the ICP-MS measurement technique and the many potential sources of interferences observed when determining the most common metals found in vaping devices. The series will wrap up by posing some questions to state regulators as to how the data could be used to regulate cannabis vaping devices.

For a detailed overview of how testing of ENDS is achieved, you can find a talk from Dr Steven Pappas, Tobacco Inorganics Group, CDC available to view for free here. 

Further reading

1. ISO Method, 20768:2018, Vapor Products - Routine Analytical Vaping Machine - Definitions and Standard Conditions, https://webstore.ansi.org/Standards/ISO/ISO207682018?gclid=EAIaIQobChMI2r70-6Ls7AIVHOy1Ch329wcKEAAYASAAEgIQA_D_BwE 
2. CORESTA Recommended Method Number 81, Routine Analytical Machine for E-Cigarette Aerosol Generation and Collection – Definitions and Standard Conditions, June, 2015, https://www.coresta.org/sites/default/files/technical_documents/main/CRM_81.pdf 
3. Cerulean CETI 8 E-cigarette vaping instrument (8 Channel), https://www.cerulean.com/en/solutions/product/ceti-8-range 
4. Analysis of Toxic Metals in Electronic Cigarette Aerosols Using a Novel Trap Design, M. Halstead et.al., Journal of Analytical Toxicology, 2020;44:149–155. 
5. Measurement of Elemental Constituents of Vaping Liquids and Aerosols with ICP-MS. R.S. Pappas et al. in R.J. Thomas, Measuring Heavy Metal Contaminants in Cannabis and Hemp, CRC Press, 2020, ISBN 9780367417376.
6. The Status of US States’ Legalization of Medical Marijuana,  https://medicalmarijuana.procon.org/legal-medical-marijuana-states-and-dc/ 
7. Regulating Heavy Metal Contaminants in Cannabis: What Can be Learned from the Pharmaceutical Industry? R. J. Thomas, Analytical Cannabis, June 30, 2020, https://www.analyticalcannabis.com/articles/regulating-heavy-metal-contaminants-in-cannabis-what-can-be-learned-from-the-pharmaceutical-312494 
8. United States Pharmacopeia General Chapter  <232>  Elemental Impurities – Limits: First Supplement to USP 40–NF 35, 2017, https://www.usp.org/chemical-medicines/elemental-impurities-updates 
9. The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), Q3D, (R1) – Guideline for Elemental Impurities, ICH Website: https://www.ich.org/page/quality-guidelines#3-6 
10. Toxic Metal-Containing Particles In Aerosols from Pod-Type Electronic Cigarettes. R.S Pappas, et al., Journal of Analytical Toxicology, 2020; doi: 10.1093/jat/bkaa088.