Simultaneous determination of nicotine, cotinine, norcotinine, and trans-3′-hydroxycotinine in human oral fluid using solid phase extraction and gas chromatography–mass spectrometry

Nicotine is rapidly and extensively metabolized in humans. We present an analytical method to simultaneously quantify nicotine, cotinine, norcotinine, and trans-3′-hydroxycotinine in human oral fluid. Solid phase extraction (SPE) and GC/MS/EI with selected ion monitoring (SIM) were utilized. Linearity ranged from 5 to 1000 ng/mL of oral fluid; correlation coefficients for calibration curves were >0.99. Recoveries were 90–115% nicotine, 76–117% cotinine, 88–101% norcotinine, and 67–77% trans-3′-hydroxycotinine. Intra-assay precision and accuracy ranged from 1.6 to 5.7% and 1.6 to 17.8%, respectively. Inter-assay precision and accuracy ranged from 4.3 to 10.2% and 0 to 12.8%, respectively. Suitable precision and accuracy were achieved for the simultaneous determination of nicotine and three metabolites in the oral fluid of smokers. This assay is applicable to pharmacokinetic studies of nicotine, cotinine, and trans-3′-hydroxycotinine from tobacco smokers and can be utilized for routine monitoring of tobacco smoke exposure. 3-Hydroxycotinine requires additional investigation to determine its usefulness as a biomarker for tobacco smoke exposure.