Abstract: To separate S-(-)-nicotine and R-(+)-nicotine in e-liquids, a reversed-phase HPLC-DAD method coupled with a chiral column was developed. The sensory differences between the two nicotine isomers were studied. The results showed that: 1) The optimized chromatography condition included an Agilent InfinityLab Poroshell Chiral-T column, with a mobile phase consisted of methanol and acetonitrile at the volume ratio 45?55 (containing 0.25% acetic acid and 0.05% trimethylamine in methanol), and the flow rate of 1 mL/min under isocratic elution. 2) S-(-)-nicotine and R-(+)-nicotine presented good linear relationships, and the limits of detection (LODs) were both 0.008 mg/g. The intra- and inter-day RSDs of S-(-)-nicotine were 0.89% and 1.26%, and those of R-(+)-nicotine were 3.98% and 4.70%, respectively. The recoveries of S-(-)-nicotine and R-(+)-nicotine were in the range of 95.87%-104.22% and 102.43%-108.85% respectively. 3) The contents of S-(-)-nicotine and R-(+)-nicotine in 20 e-liquid samples available in Chinese markets were determined. S-(-)-nicotine was the main isomer with the contents ranging from 2.325 to 22.042 mg/g. The optical purities of S-(-)-nicotine was in the range of 96.74%-100%, and no racemic nicotine was found. 4) The strength, satisfaction and irritation of S-(-)-nicotine were the strongest; those of R-(+)-nicotine were the lowest; while those of (R, S)-nicotine were in between.