Performance Evaluation of a Nontargeted Platform Using Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry Integrating Computer-Assisted Structure Identification and Automated Semiquantification for the Comprehensive Chemical Characterization of a Complex Matrix

Abstract

Nontargeted screening methodologies are powerful approaches for comprehensive chemical characterization of complex matrixes. In order to maximize chemical space coverage, three analytical methods using two-dimensional gas chromatography with time-of-flight mass spectrometry for nonpolar, polar, and volatile compounds have been established. The structural identification process was streamlined with an in-house developed computer-assisted structure identification platform, which facilitated the identification of novel compounds and also delivered semiquantitative concentrations for all compounds. Key performance parameters for this nontargeted platform, including chemical space coverage, confidence for structural identification, accuracy of semiquantification, and performance of differential analysis, were evaluated. The automated structural identification process was assessed using a subset of 243 compounds (out of 2990), which were confirmed to be present in cigarette smoke using reference standards. Consistently high true positive identification rates between 88.2% and 96.2% across the different concentration ranges investigated were demonstrated. Accuracy for semiquantification was assessed by comparison with quantitative data from literature, where a maximum 4-fold deviation from available targeted analysis values was estimated.

Comprehensive chemical characterization of the aerosol generated by a heated tobacco product by untargeted screening

“…During this method evaluation process, it was observed that the number of compounds identified to be present in the smoke of the 3R4F reference cigarette increased exponentially as their concentration decreased. By extrapolating this exponential trend and comparing the anticipated number versus the actual number of compounds identified, a point was reached at a concentration of 100 ng/item, below which the actual number of compounds identified was consistently lower than expected, which was interpreted as being the lower limit of the working range for the methodology [18]. While the number of compounds increased as their concentrations decreased, the mass contribution relative to the total chemical space identified declined logarithmically and the accumulated total mass for compounds below 100 ng/item for 3R4F was estimated to be less than 1% of the total mass amenable by GC×GC-TOFMS analysis [18].…”

“…By extrapolating this exponential trend and comparing the anticipated number versus the actual number of compounds identified, a point was reached at a concentration of 100 ng/item, below which the actual number of compounds identified was consistently lower than expected, which was interpreted as being the lower limit of the working range for the methodology [18]. While the number of compounds increased as their concentrations decreased, the mass contribution relative to the total chemical space identified declined logarithmically and the accumulated total mass for compounds below 100 ng/item for 3R4F was estimated to be less than 1% of the total mass amenable by GC×GC-TOFMS analysis [18]. A formal performance assessment specific for the LC-HRAM-MS methods has not yet been performed; however, the characteristics were assumed to be broadly comparable, and a 100 ng/item threshold was applied to data generated by both GC×GC-TOFMS and LC-HRAM-MS platforms, which served to optimize the proportion of substances identified relative to a practicable amount of effort applied for their identification.…”

“…1). These methods are based on comprehensive two-dimensional gas chromatography with time-offlight mass spectrometry (GC×GC-TOFMS) [18] and liquid chromatography with high-resolution accurate mass spectrometry (LC-HRAM-MS) [19]. They are semi-quantitative in nature, whereby the concentrations of a large number of compounds are estimated versus a limited number of stable isotope-labeled reference standards of known concentrations, using an internal standardization approach.…”

“…For constituents amenable to gas chromatographic analysis, quantitative values derived from analysis of recognized HPHCs by using targeted methods were compared with corresponding semi-quantified values from untargeted analysis. This demonstrated that, for analytes that were measureable by both techniques, the untargeted approach using GC×GC-TOFMS was able to semi-quantify within a ± 4-fold deviation from the true concentration [18]. Available liquid chromatography-based targeted methods were insufficient to make a similar assessment for LC-HRAM-MS.…”

“…The number of accumulations collected was optimized to match the requirements for individual analytical methods, and replicate sample collection was performed for each required measurement (at least triplicate). The samples were subsequently analyzed by GC×GC-TOFMS [18] and LC-HRAM-MS [19].…”