Nab d4

All samples of the four TSNAs (N′-nitrosonornicotine, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, N′-nitrosoanatabine, and N′-nitrosoanabasine) were purchased from Merck (Darmstadt, Germany), while the deuterated samples (NNK-d4, NNN-d4, NAT-d4, and NAB-d4) were obtained from Toronto Research Chemicals (Toronto, Canada). All solvents used were of analytical grade (ACN, MeOH, acetic and formic acids, ammonia, ethyl acetate, and methylene chloride) and were purchased from Merck (Darmstadt, Germany). The sheets of PP membrane (200 × 200mm, 0.1 µm) were purchased from GVS Filter Technology (Lancaster, United Kingdom). Ultrapure water was prepared by an HLP5 system from Hydrolab (Wiślina, Poland), while PG (99.8%) and VG (99.5%) were obtained from Anwit (Warsaw, Poland). Individual stock solutions of analytes and their corresponding IS used in this study were prepared by dissolving appropriate amounts in the ACN solvent to obtain 40 µg/mL of each. Working standard mixtures were obtained by dilution in the mobile phase. Calibration solutions were prepared by adding the appropriate amount to obtain 1, 5, 10, 20, 50, and 75 ng/g of each analyte, while the IS concentration was kept at 20 ng/g of each. All solutions were stored in a fridge at 4 °C for up to 4 weeks.

Five standard TSNAs, NNK, NNN, NAT, NAB and NNAL, and their stable isotope-labeled compounds, NNK-d₃ (isotopic purity 99.9%), NNN-d₄ (isotopic purity 97.8%), NAT-d₄ (isotopic purity 98.0%), NAB-d₄ (isotopic purity 99.7%) and NNAL-d₅ (isotopic purity 97.8%) as each internal standard (IS) (supplementary Figure S3), were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Stock solutions of 1.0 mg mL^–1 of each compound were prepared by dissolving in LC–MS grade acetonitrile and diluted with distilled water to the required concentration prior to use. The mixed standard solution consisted of 1 ng mL^–1 each of NNK, NNN, NAT and NAB and 10 ng mL^–1 NNAL, whereas the mixed IS solution consisted of 0.1 ng mL^–1 each of NNK-d₃, NNN-d₄, NAT-d₄ and NAB-d₄, and 1 ng mL^–1 NNAL-d₅. These prepared solutions were stored at 4℃. Methanol and distilled water as mobile phases were of LC–MS grade, while all other chemicals were of analytical reagent grade.

Water (H₂O), methanol (MeOH), dichloromethane (DCM), pentane (Pent), hexane, isopropyl alcohol (IPA), ethyl acetate (EtAc), acetone, toluene, butanol (HPLC grade), sulfuric acid (H₂SO₄), hydrochloric acid (HCl), ammonium hydroxide (NH₄OH), potassium carbonate (K₂CO₃), tetrasodium EDTA (Na₄EDTA), and ammonium formate (AmFor) (reagent grade) were purchased from Fisher Scientific (USA).
Nicotine base was purchased from Sigma Aldrich (USA). Native nicotine and nicotine-d₄ salts were synthesized as described in Ref.^{[30 ]}. Nicotelline and deuterated nicotelline-d₈ were synthesized as previously reported^{[16 (link)]}. Deuterated nitrosamines NNK-d₄, NNN-d₄, NAT-d₄, and NAB-d₄ and native nitrosamines NNK, NNN, NAT, and NAB were purchased from Toronto Research Chemicals (Canada). The native PAH QTM mix and chrysene-d₁₂, were purchased from Supelco (USA) and SPEXCertiPrep (USA), respectively. Deuterated PAH were purchased from Chemservice (USA). Agilent Bond Elut 10 mL and 1 mg silica column were purchased from Agilent (USA).