Simultaneous quantification of tobacco alkaloids and major phase I metabolites by LC-MS/MS in human tissue.

INTRODUCTION: Insurance agencies might request laboratories to differentiate whether a deceased has been a smoker or not to decide about refunding of his nonsmoker rate. In this context, the question on a solid proof of tobacco alkaloids and major metabolites in tissues came up. Currently, an appropriate assay is still lacking to analyze tissue distribution in smokers or nonsmokers. Nicotine (NIC), nornicotine (NNIC), anatabine (ATB), anabasine (ABS), and myosmine (MYO) are naturally occurring alkaloids of the tobacco plant; most important phase I metabolites of NIC are cotinine (COT), norcotinine (NCOT), trans-3'-hydroxycotinine (HCOT), nicotine-N'-oxide (NNO), and cotinine-N-oxide (CNO). An analytical assay for their determination was developed and applied to five randomly selected autopsy cases. METHODS: Homogenates using 500 mg aliquots of tissue samples were analyzed by liquid chromatography/tandem mass spectrometry following solid phase extraction. The method was validated according to current international guidelines. RESULTS: NIC, COT, NCOT, ABS, ATB, and HCOT could be detected in all tissues under investigation. Highest NIC concentrations were observed in the lungs, whereas highest COT concentrations have been found in the liver. MYO was not detectable in any of the tissues under investigation. CONCLUSIONS: The assay is able to adequately separate isobaric analyte pairs such as NIC/ABS/NCOT and HCOT/CNO thus being suitable for the determination of tobacco alkaloids and their phase I metabolites from tissue. More autopsy cases as well as corresponding body fluids and hair samples will be investigated to differentiate smokers from nonsmokers.