Simultaneous quantification of nicotine, cotinine, trans-3′-hydroxycotinine, norcotinine and mecamylamine in human urine by liquid chromatography–tandem mass spectrometry

Abstract

BackgroundMecamylamine is a nicotine antagonist under investigation in combination with nicotine replacement for smoking treatment. MethodsA simple, rapid and reliable liquid chromatography tandem mass spectrometry (LCMSMS) method was developed and validated for quantifying nicotine, cotinine, trans-3′-hydroxycotinine, norcotinine and mecamylamine in human urine. Chromatography was performed on a Synergi PolarRP column with a gradient of 0.1% formic acid and 0.1% formic acid in acetonitrile at 0.25ml/min with an 8-min total runtime. Analytes were monitored by positive mode electrospray ionization and multiple reaction monitoring mass spectrometry. ResultsLinear dynamic ranges were 1–500ng/ml for nicotine and norcotinine, 0.5–500ng/ml for trans-3′-hydroxycotinine, 0.2–500ng/ml for cotinine, and 0.1–100ng/ml for mecamylamine; correlation coefficients were consistently greater than 0.99, and all calibrator concentrations were within 20% of target. Extensive endogenous and exogenous interferences were evaluated. At 3 concentrations spanning the linear dynamic range of the assay, mean extraction efficiencies from urine were 55.1–109.1% with analytical recovery (bias) 82.0–118.7% and total imprecision of 0.7–9.1%. Analytes were stable for 24h at room temperature, 72h at 4°C, 72h in autosampler at 15°C and after three freeze/thaw cycles. ConclusionThis method is useful for monitoring mecamylamine, nicotine and nicotine metabolites in smoking cessation and other clinical nicotine research.