Simultaneous and Sensitive Measurement of Anabasine, Nicotine, and Nicotine Metabolites in Human Urine by Liquid Chromatography–Tandem Mass Spectrometry

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Abstract
Background: Determination of nicotine metabolism/pharmacokinetics provides a useful tool for estimating uptake of nicotine and tobacco-related toxicants, for understanding the pharmacologic effects of nicotine and nicotine addiction, and for optimizing nicotine dependency treatment. Methods: We developed a sensitive method for analysis of nicotine and five major nicotine metabolites, including cotinine, trans-3′-hydroxycotinine, nicotine-N′-oxide, cotinine-N-oxide, and nornicotine, in human urine by liquid chromatography coupled with a TSQ Quantum triple quadrupole tandem mass spectrometer (LC/MS/MS). Urine samples to which deuterium-labeled internal standards had been added were extracted with a simple solid-phase extraction procedure. Anabasine, a minor tobacco alkaloid, was also included. Results: The quantification limits of the method were 0.1–0.2 μg/L, except for nicotine (1 μg/L). Cotinine-N-oxide, trans-3′-hydroxycotinine, nicotine, and anabasine in urine were almost completely recovered by the solid-phase extraction, whereas the mean extraction recoveries of nicotine-N′-oxide, cotinine, and nornicotine were 51.4%, 78.6%, and 78.8%, respectively. This procedure provided a linearity of three to four orders of magnitude for the target analytes: 0.2–400 μg/L for nicotine-N′-oxide, cotinine-N-oxide, and anabasine; 0.2–4000 μg/L for cotinine, nornicotine, and trans-3′-hydroxycotinine; and 1.0–4000 μg/L for nicotine. The overall interday method imprecision and recovery were 2.5–18% and 92–109%, respectively. Conclusions: This sensitive LC/MS/MS procedure can be used to determine nicotine metabolism profiles of smokers, people during nicotine replacement therapy, and passively exposed nonsmokers. This method avoids the need for a time-consuming and labor-intensive sample enrichment step and thus allows for high-throughput sample preparation and automation.

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