A rapid high pressure liquid chromatography coupled with tandem mass spectrometry method (LC-MS/MS) was developed for simultaneously determining nicotine (NT) and its derivatives including cotinine (CT), trans-3-hydroxycotinine (HC), nornicotine (NNT), anatabine (AT), anabasine (AB) and amino ketone (4-(methylamino)-1-(3-pyridyl)1-butanone) (AK) as well as two important metabolites isomers 2-hydroxynicotine (2-HN) and 6-hydroxynicotine (6-HN) in tobacco leaves. Different from other methods, 2-HN and 6-HN isomers can be successfully separated and quantified through a combination of amide and C18 columns and optimized multiple reaction monitoring mode of tandem mass spectrometry. The limits of detections (LOD) and quantification (LOQ) of these compounds were 0.02 and 0.06 ng/mL for CT and HC, 0.05 and 0.15 ng/mL for AK and 6-HN, and 0.1 and 0.3 ng/mL for AB, NNT, AT, 2-HN and NT, respectively. Their recoveries ranged from 86.1% for 2-HN to 102.2% for AK by using solvent extraction assisted with ultrasonication. The developed method was validated through determination of the compounds in different regular and genetically modified (for lowering nicotine) tobacco leaves. Conclusively, it is a robust and rapid method for simultaneous quantification of nicotine and its derivatives in tobacco products. The protocol of the method could also be applied in developing analysis method for tracking the toxic substances synthesis and metabolism in tobacco leaves or other herbal plants.

开发了一种快速高压液相色谱联用串联质谱法 (LC-MS/MS)，用于同时测定尼古丁 (NT) 及其衍生物，包括可替宁 (CT)、反式-3-羟基可替宁 (HC)、降烟碱 (NNT)、安那他滨 (AT)、安那巴辛 (AB) 和氨基酮 (4-(甲基氨基)-1-(3-吡啶基)1-丁酮) (AK) 以及两种烟叶中重要的代谢物异构体 2-羟基尼古丁 (2-HN) 和 6-羟基尼古丁 (6-HN)。与其他方法不同，2-HN和6-HN异构体可以通过酰胺和C18色谱柱的组合以及优化的串联质谱多反应监测模式进行成功的分离和定量。这些化合物的检测限 (LOD) 和定量 (LOQ) 对于 CT 和 HC，分别为 0.02 和 0.06 ng/mL，对于 AK 和 6-HN，分别为 0.05 和 0.15 ng/mL，对于 AB，分别为 0.1 和 0.3 ng/mL，分别为 NNT、AT、2-HN 和 NT。通过使用溶剂萃取辅助超声处理，其回收率从 2-HN 的 86.1% 到 AK 的 102.2% 不等。通过测定不同的常规和转基因（用于降低尼古丁）烟叶中的化合物，对开发的方法进行了验证。总之，它是一种稳健且快速的方法，可用于同时量化烟草产品中的尼古丁及其衍生物。该方法的方案还可用于开发跟踪烟叶或其他草药植物中有毒物质合成和代谢的分析方法。