Determination of nicotine and cotinine in rat plasma by liquid chromatography–tandem mass spectrometry

A method was developed for the efficient determination of nicotine and cotinine in rat plasma samples originating from nicotine exposure studies. Nicotine and cotinine were extracted from plasma samples with dichloromethane and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of hydrochloric acid to the organic solvent during evaporation. The samples were analysed using liquid chromatography with triple quadrupole mass spectrometry. For quantification, the deuterated internal standards were added and the most intensive MS–MS ion of the analyte and internal standards were monitored. For confirmatory analysis, two specific MS–MS ions, viz. m/z 132 and 106 for nicotine and m/z 80 and 98 for cotinine, were monitored and the ratios between the ions were calculated and compared with those of standards. The ratios have to be within the tolerances of the EU criteria. The limit of identification of the developed method was 1 μg/l. The repeatability ranged from 5 to 12% (R.S.D.) for nicotine and from 3 to 5% for cotinine at the concentration level of 1–60 μg/l (n=4).