A validated method for the determination of nicotine, cotinine, trans‐3′‐hydroxycotinine, and norcotinine in human plasma using solid‐phase extraction and liquid chromatography‐atmospheric pressure chemical ionization‐mass spectrometry

Abstract A liquid chromatographic‐mass spectrometric method for the simultaneous determination of nicotine, cotinine, trans ‐3′‐hydroxycotinine, and norcotinine in human plasma was developed and validated. Analytes and deuterated internal standards were extracted from human plasma using solid‐phase extraction and analyzed by liquid chromatography/atmospheric pressure chemical ionization‐mass spectrometric detection with selected ion monitoring (SIM). Limits of detection and quantification were 1.0 and 2.5 ng/ml, respectively, for all analytes. Linearity ranged from 2.5 to 500 ng/ml of human plasma using a weighting factor of 1/ x ; correlation coefficients for the calibration curves were > 0.99. Intra‐ and inter‐assay precision and accuracy were < 15.0%. Recoveries were 108.2–110.8% nicotine, 95.8–108.7% cotinine, 90.5–99.5% trans ‐3′‐hydroxycotinine, and 99.5–109.5% norcotinine. The method was also partially validated in bovine serum, owing to the difficulty of obtaining nicotine‐free human plasma for the preparation of calibrators and quality control (QC) samples. This method proved to be robust and accurate for the quantification of nicotine, cotinine, trans ‐3′‐hydroxycotinine, and norcotinine in human plasma collected in clinical studies of acute nicotine effects on brain activity and on the development of neonates of maternal smokers. Copyright © 2006 John Wiley & Sons, Ltd.