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

Mecamylamine is a nicotine antagonist under investigation in combination with nicotine replacement for smoking treatment. A 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 0.25 ml/min with an 8-min total runtime. Analytes were monitored by positive mode electrospray ionization and multiple reaction monitoring mass spectrometry. Linear dynamic ranges were 1-500 ng/ml for nicotine and norcotinine, 0.5-500 ng/ml for trans-3'-hydroxycotinine, 0.2-500 ng/ml for cotinine, and 0.1-100 ng/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, 72 h at 4 °C, 72 h in autosampler at 15 °C and after three freeze/thaw cycles. This method is useful for monitoring mecamylamine, nicotine and nicotine metabolites in smoking cessation and other clinical nicotine research.