Optimization and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, cotinine, trans-3'-hydroxycotinine and norcotinine in human oral fluid

An analytical procedure was developed and validated for the simultaneous identification and quantification of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in 0.5 mL of human oral fluid collected with the Quantisal (TM) oral fluid collection device. Solid phase extraction and liquid chromatography-tandem mass spectrometry with multiple reaction monitoring were utilized. Endogenous and exogenous interferences were extensively evaluated. Limits of quantification were empirically identified by decreasing analyte concentrations. Linearity was from 1 to 2,000 ng/mL for nicotine and norcotinine, 0.5 to 2,000 ng/mL for trans-3'-hydroxycotinine, and 0.2 to 2,000 ng/mL for cotinine. Correlation coefficients for calibration curves were > 0.99 and analytes quantified within +/- 13% of target at all calibrator concentrations. Suitable analytical recovery (> 91%) was achieved with extraction efficiencies > 56% and matrix effects < 29%. This assay will be applied to the quantification of nicotine and metabolites in oral fluid in a clinical study determining the most appropriate nicotine biomarker concentrations differentiating active, passive, and environmental nicotine exposure.