untargeted assessment strategy

      Untargeted assessment in the development of smoke-free products

      In order to ensure that the use of new technologies (such as heat-not-burn) does not introduce any new risk to adult smokers when compared to the use of cigarettes, we perform analyses to determine whether any new or unexpected chemical compounds are present in the aerosol of our smoke-free products.

      In addition to verifying the reduction of established toxicants released by the Tobacco Heating System (THS) compared with cigarettes, we have also adopted a strategy for the comprehensive, untargeted screening of smoke-free product aerosols. Quantitative analysis targets specific harmful and potentially harmful constituents (HPHCs) of interest to the exclusion of all others, while our untargeted approach aims to identify any chemicals which may be present in the aerosol.

      Our untargeted assessment involves the use of chromatographic separation coupled with high-resolution mass spectrometry. Since the chemical diversity of constituents within an aerosol is so large, a number of overlapping methods are required to ensure comprehensive analytical coverage of constituent polarities, volatilities, and structural classes. Our research involves both gas and liquid chromatography combined with mass spectrometry to separate the chemicals and identify them by weight.


      Chromatographic separation and mass spectrometry

      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.

      The impact of combustion on aerosol constituents

      In a later study, we conducted a similar comparison between THS and cigarettes which were produced from the same blend of tobacco and not adding flavorants in order to minimize confounding factors, allowing a clearer focus on the differences due to heating versus burning tobacco. Among the peaks identified in the chromatograph of the aerosol, only 3.5%—corresponding to 31 distinctive compounds—were significantly more abundant in THS aerosol than in cigarette smoke, with a notable subset of the compounds identified as reaction products of glycerol. These results demonstrate that heating a glycerol-containing tobacco substrate to the temperatures applied in THS does not introduce new compounds in the resulting aerosol compared to CC smoke which are detectable with the method portfolio applied in this study.  

      This research was published in 2024 in a study titled “Non-targeted analytical comparison of a heated tobacco product aerosol against mainstream cigarette smoke: does heating tobacco produce an inherently different set of aerosol constituents?” in the journal Analytical and Bioanalytical Chemistry.

      Untargeted screening

      Learn more about our comprehensive chemical characterization

      In addition to the methods described above, you can find a more detailed summary of this research and the results in the following resources: