For each method, an array of retention index markers is included in order to improve the speed and accuracy of compound identification. In addition, a number of stable isotope-labeled internal standards are included in order to provide a semi-quantitative estimate of constituent abundance. Compounds are identified using an automated software platform developed in-house, which interrogates both commercially available and custom-built mass spectral databases and compares a number of predicted versus measured parameters, such as relative retention time, to improve the certainty for identification.
When developing smoke-free products or creating subsequent iterations of an existing smoke-free product, it is important to understand whether any new compounds are generated in comparison to a reference product or previous version. It is also important to identify whether any significant increase in the concentration of a common constituent has occurred. In this situation, untargeted differential screening is performed, whereby data from the untargeted screening of different products are compared. Statistical models are used to filter compounds with significant differences, followed by a ranking procedure which considers the relative differences in abundance of each compound as well as the absolute abundance. The findings are then passed on for toxicological assessment.
Untargeted chemical profiling
Our analysis found that the total particulate matter (TPM) of the THS aerosol contains more than 90% water, glycerin, and nicotine. The residual material was characterized using untargeted screening methods. In cigarette smoke, water, glycerin, and nicotine make up less than half of the TPM.
An important part of this untargeted chemical profiling is the accurate identification of the compounds that are detected. This is achieved by comparing the mass spectral data with reference libraries, aided by computer-assisted structure identification tools as described above.
Wherever possible, the proposed identities of the compounds are confirmed using reference standards. Using this approach, we identified a total of 529 compounds that were present at greater than 100 ng in the aerosol produced by one representative THS tobacco stick variant, excluding water, glycerin, and nicotine.
We confirmed the identities of 80% of the compounds in THS aerosol, representing more than 96% of the total mass of the aerosol, using reference standards. All 529 compounds were also found in the smoke of a 3R4F reference cigarette.
Untargeted differential screening
We next aimed to compare the relative abundance of compounds in THS aerosol and cigarette smoke by untargeted differential profiling. The approach can highlight compounds that are unique to the aerosol, or more abundant in it than in smoke, even if these compounds are below 100 ng/stick. Using this approach, we detected 51 compounds that were more abundant in the aerosol of a representative THS tobacco stick variant than in smoke from 3R4F reference cigarettes.
We also detected three compounds unique to the aerosol. These three compounds are not considered to be of toxicological concern at the low levels measured in THS aerosol. In addition, toxicological evaluation of the 51 more abundant aerosol constituents highlighted four compounds classified as carcinogens and/or mutagens. Although the compounds might have an impact on the toxicological activity of THS aerosol, in vitro and in vivo toxicological investigations, including genotoxicity endpoints, have been performed on the THS aerosol and showed overall decreased toxicity compared with cigarettes.
This research was published in 2020 in a study titled "Comprehensive chemical characterization of the aerosol generated by a heated tobacco product by untargeted screening” in the journal Analytical and Bioanalytical Chemistry.
