Method validation of non-targeted analysis of mainstream smoke constituents from the 1R6F reference cigarette

In this study, we developed a systematic validation framework for non-targeted analysis (NTA) and applied it to complementary analytical workflows using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS) and liquid chromatography-trapped ion mobility spectrometry-quadrupole time-of-flight mass spectrometry (LC-TIMS-QTOFMS) for broad profiling of cigarette mainstream smoke constituents. The validation framework evaluated instrumental detection and annotation performance, method-specific library-search thresholds, the accuracy of semi-quantitative concentration estimates, repeatability and intermediate precision, and analytical coverage across two GC × GC-TOFMS methods and three LC-TIMS-QTOFMS methods. An operational reporting threshold of 100 ng per cigarette was selected as the lowest evaluated level at which the mean true-positive rate across the analytical methods was at least 90% and no individual method showed a true-positive rate below 80%. The method-specific library-similarity-score thresholds used to define high-confidence annotations ranged from 700 to 800. For the GC × GC-TOFMS methods, the accuracy of semi-quantitative concentration estimates for the quality control (QC) compounds ranged from 41% to 128%. In the LC-TIMS-QTOFMS methods, the initial semi-quantitative concentration estimates showed substantial compound-dependent positive bias; application of predicted relative response factors reduced this bias, although the accuracy of the adjusted semi-quantitative estimates still ranged from 67% to 428%. The coefficients of variation for repeatability and intermediate precision were below 30% for all evaluated QC compounds across all analytical methods. The summed semi-quantitative concentration estimates obtained using the NTA workflows, together with separately determined amounts of major constituents, including nicotine, water, propylene glycol, glycerin, and triacetin, represented 39.6 mg/cig. This amount corresponded to an apparent mass ratio of 91.0% relative to the gravimetrically determined total particulate matter (TPM) yield. The annotated constituents also demonstrated broad and complementary chemical space coverage in terms of molecular weight and calculated log P. Application of the evaluated workflows to mainstream smoke from the Kentucky reference cigarette (1R6F) resulted in 3590 reported analytical features after consolidation of duplicate "Confirmed" identifications across analytical methods. These results demonstrate that the proposed framework provides a systematic basis for evaluating and further developing NTA workflows for cigarette smoke and other complex chemical mixtures.