Fast and sample cleanup-free measurement of nicotine and cotinine by stable isotope dilution ultra-performance liquid chromatography-tandem mass spectrometry

Abstract

We developed a stable isotope dilution ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay to measure nicotine and cotinine, the major oxidative and pharmacologically less active metabolite of nicotine, in human urine. A simple dilution step was used as sample preparation and the measurement of nicotine and cotinine was performed during a 1.5-min run-time using nicotine-D₄ and cotinine-D₄ as internal standards. Multiple calibration curves for the analysis of both nicotine and cotinine exhibited a consistent excellent linearity and reproducibility in the range of 5-35,000 μg/L (r>0.999). Limits of Detection were 0.7 μg/L for nicotine and 0.4 μg/L for cotinine, and Lower Limits of Quantification were 1.7 μg/L for nicotine and 1.1 μg/L for cotinine. The intraassay coefficients of variation (CVs) for nicotine and cotinine were <4% and <2%, respectively, the interassay CVs were <6% for nicotine and <4% for cotinine. The inaccuracy was <6% for both substances. The mean recovery was 103.2% (range 96.8-105.1%) for nicotine and 97.4% (range 94.3-99.2%) for cotinine. A method comparison showed that the values of nicotine metabolites in human urine samples (n=98) measured by a commercially available chemiluminescent immunoassay tested on analyzer IMMULITE 2000 were much higher than the cotinine concentration in the same urine samples measured by our UPLC-MS/MS assay. The Passing-Bablok regression line was: immunoassay=4.62 (UPLC-MS/MS)+3.64 [μg/L]; r=0.75. This robust, sensitive and interference-free UPLC-MS/MS assay permits rapid and accurate determination of nicotine and cotinine in human urine.