Quantifying Tobacco Alkaloids and Nitrosamines in Dust

Concentrations of tobacco alkaloids and tobacco-specific nitrosamines were determined using previously described analytical protocols^{7 (link), 8 (link)}, with a modified extraction procedure, described below and in the Supporting Information, Figure S1. Each dust sample was homogenized and fractionated using a mechanical shaker equipped with a 100-mesh sieve to obtain dust particles smaller than 150 μm. Fine dust samples were accurately weighed (66 ± 11 mg) into 16x125 mm culture tubes and to each sample was added 150 µL of aqueous internal standard solution (containing d₄-nicotine, d₄-NNN, d₄-NNK, d₃-NNA, d₄-NAB, d₄-NAT, d₉-cotinine, d₄-myosmine, d₄-N-formylnornicotine, d₈-nicotelline, and d₄-2,3’-bipyridine; for details see Hang et al.^{8 (link)}), 1.5 mL distilled water, and 0.5 mL of 1M sulfuric acid. The mixture was vortexted and 10 mL of 70:30 toluene:butanol was added. The tubes were then sonicated at 55°C for 1 hour with intermittent vortexing. The tubes were centrifuged, frozen in a dry ice-acetone bath and the toluene:butanol phase discarded. After the remaining aqueous phase was made basic with 1 mL of 45% potassium carbonate 5% tetrasodium EDTA, 10 mL of 45:45:10 dichloromethane:pentane:ethyl acetate was added. The samples were extracted using vortex mixing, centrifuged, and frozen again in a dry ice-acetone bath and the organic layer divided into two sets of 13x100 mm culture tubes for analysis by GC-MS (Agilent 6890N, for nicotine) and liquid chromatography-tandem mass spectrometry (Thermo Fisher Vantage LC-MS/MS, for all other analytes), as previously described.^{7 (link), 8 (link)} Representative LC-MS/MS chromatograms are provided in the Supporting Information, Figures S2 and S3. Concentrations were calculated using the instrument data system software, aqueous standards spanning the measured concentration range, and calibration curves prepared from analyte/internal standard peak area ratios and analyte concentrations using linear regression with 1/X weighting. The precision of the analytical method was demonstrated using National Institute of Standards and Technology Standard Reference Material 2585 (Organic Contaminants in House Dust). Table S1 (Supporting Information) shows coefficients of variation for each analyte in nine analytical replicates of three extracts of the standard reference material ranging in sample mass from 31 to 110 mg.