Abstract
A rapid, accurate, and reproducible method was developed for high-throughput testing of nicotine, cotinine, trans-3'-hydroxycotinine, nornicotine, norcotinine, and anabasine in urine. Data show that a fast and highly efficient analysis of these basic compounds can be achieved with the Raptor™ Biphenyl column using standard low-pH, reversed-phase LC-MS mobile phases that are compatible with a variety of LC-MS instrumentation.
Introduction
Nicotine is the major tobacco alkaloid that underlies addiction in tobacco users. Anabasine and anatabine are the most abundant minor alkaloids in tobacco [1]. Anabasine is frequently used as a unique marker for recent tobacco use as it can only be detected in the urine of tobacco users and is not present in the urine of those who use nicotine replacement therapies (e.g., nicotine patches). In humans, more than 70% of nicotine is transformed to cotinine, which is subsequently converted to trans-3'-hydroxycotinine, the main nicotine metabolite detected in urine [2]. Nornicotine and norcotinine are minor metabolites (0.5–2%) produced by the demethylation of nicotine and cotinine, respectively. The urinary measurement of nicotine metabolites has several applications, including public tobacco exposure monitoring, nicotine replacement therapy evaluation, drug therapy assessment, forensic toxicology analysis, and life or health insurance application. In addition, nicotine metabolites can be used as the biomarkers for pharmacogenomics evaluation and disease profiling [3]. A variety of chromatographic methods have been developed for nicotine metabolite analysis. However, most methods use high-pH chromatography with relatively high concentrations of additives to increase retention, improve peak shape, and reduce peak tailing. The intent of this application was to develop a method for the analysis of nicotine-related compounds in urine using solutions that are “friendly” to LC-MS/MS systems. A Raptor™ Biphenyl column was chosen as the analytical column because it provides good retention and peak shape for the target analytes when used with standard low-pH, reversed-phase mobile phases. The clinical applicability of this method was demonstrated by the accurate and reproducible analysis of fortified analytes in urine.
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