Abstract: In order to investigate the transfer behavior of nicotine isomers between heated tobacco products and combustible cigarettes in mainstream smoke, a reversed-phase two-dimensional liquid chromatography method based on trap column capture and heart cutting techniques was developed for separating and analyzing S-(-)-nicotine and R-(+)-nicotine isomers in cigarette smoke and heated tobacco product aerosol. The developed method was used to study the differences of R-(+)-nicotine to S-(-)-nicotine ratio (shorted as R /S ratio) in cigarette smoke and heated tobacco product aerosol under Health Canada Intense smoking regime. The results showed that: 1) The R/S ratio in tobacco of cigarette was in the range of 0.16%-0.35%, which was equivalent to that of centrally heated tobacco product; however, the R/S ratio in tobacco of circumferentially heated tobacco product was obviously lower. 2) The R/S ratio in mainstream smoke of cigarette was obviously higher than that in aerosol of heated tobacco products; that of centrally heated tobacco product was higher than that of circumferentially heated tobacco product. The R/S ratios in cigarette smoke and heated tobacco product aerosol were correlated to puffing temperature.