Recently, synthetic nicotine (SN) has become commercially available and can be supplied as United States Pharmacopeia (USP) grade (S)-nicotine, as a 50/50 (S)/(R) mixture, or as a mixture in varying ratios of (S)/(R) enantiomers. Some tobacco-product manufacturers may have turned to the use of SN as a replacement for tobacco-derived nicotine (TDN) in an effort to circumvent the FDA Center for Tobacco Products (CTP) Premarket Tobacco Product Application (PMTA) process for a new tobacco product or as a result of the receipt of a PMTA Marketing Denial Order. Because of recent Congressional action, the FDA now requires a PMTA for tobacco products containing SN. As tobacco product development continues, the need for robust analytical methods to differentiate TDN from SN will be of significant importance for tobacco authentication. The main purpose of this study was to demonstrate the utility of analytical chromatographic methods to distinguish TDN from SN. Nicotine samples were analysed by GC/MS and SPME/GC/MS and by either chiral GC-MS or chiral HPLC-UV. Several TDN samples were found to contain 2,3’-bipyridine, characteristic of TDN. Some SN samples were found to contain the synthetic starting material, ethyl nicotinate and the synthetic impurity, 1-methyl-2-pyrrolidinone. By chiral chromatography, SN was found to contain either a 50:50 mixture of (R)- and (S)-nicotine or a low level of (R)-nicotine (0.1-0.2%) compared to higher amounts in TDN (0.8%-0.9%). A low level of (R)-nicotine indicated that the nicotine was likely synthetic. The use of these chromatographic methods provides guidance for distinguishing TDN from SN in commercial nicotine samples.