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After incubation at 22 1C and 60 3% relative humidity for 48 h, the smoke from DNC cigarettes and 3R4F reference cigarettes (University of Kentucky Lexington, KY, USA) was generated using a smoking machine (Model SML2000, Anhui, China) in accordance with the ISO 3308 Standard Smoking procedure: puff volume 35 mL, puff duration 2 s, 60 s time interval between puffs, and no ventilation holes were blocked ( Gas chromatography-mass spectroscopy analysis of nicotine and minor alkaloids The filter disc was extracted with a 5% NaOH aqueous solution for 30 min, then triethylamine dichloromethane and an internal standard were added and mixed

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