Matrix-wise Raman threshold classification of cooling agents in e-cigarette liquids using methanol redissolution, mixed-standard robustness testing and external validation

The growing diversity of e-cigarette products creates a need for rapid analytical approaches that can screen multiple cooling agents against predefined concentration limits. Here, we developed a matrix-aware Raman workflow for threshold-oriented assessment of D,L-menthol, WS-3 and WS-23 in commercial e-cigarette liquids. Methanolic standards were first used to establish concentration-responsive Raman bands, after which analyte-specific models were developed from standard-addition measurements in real commercial matrices. Spectra were processed using a fixed preprocessing and methanol-reference normalization procedure, and predictions were original-sample-equivalent concentration before comparison with the corresponding thresholds of 6 wt% for D,L-menthol and 1 wt% for WS-3 and WS-23. A dedicated mixed-standard strategy was further used to evaluate threshold robustness under simultaneous analyte signals, spectral overlap and matrix interference. The development dataset comprised 33 unique commercial matrices, providing grouped evaluations of 26 matrices for D,L-menthol and 19 matrices each for WS-3 and WS-23. Matrix-grouped leave-one-matrix-out evaluation achieved threshold agreement of 25/26, 17/19 and 19/19, respectively. Mixed-standard robustness testing across 23 commercial matrices yielded 51/54 correct analyte-matrix decisions. An independent external set of four commercial matrices with GC-MS-quantified reference concentrations yielded 6/6 correct prespecified decisions. These results indicate that the threshold-oriented Raman strategy can provide useful discrimination of below- and above-limit samples across diverse commercial matrices. Performance remained less stable for WS-3 and some near-threshold cases, for which broader validation cohorts are warranted. Overall, the workflow shows practical potential for rapid multi-analyte exceedance prescreening before confirmatory chromatographic analysis.