Determination of nicotine exposure in dogs subjected to passive smoking using methanol extraction of hair followed by hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry

There has been no previous assessment of the level of nicotine exposure in companion animals as a result of passive smoking. A method was developed for the determination of nicotine in dog hair where extraction was carried out by sonication in methanol. The levels of nicotine obtained by extraction with methanol were found to be comparable to the lengthier method involving digestion of the hair in 1 M NaOH. The methanol extracts were injected directly onto a ZICHILIC column coupled to an Exactive high resolution Fourier Transform mass spectrometer. Endogenous nicotine was quantified against 2H4-nicotine spiked into the extraction medium, linearity was found over a wide range with the calibration curve having a slope close to 1 indicating an equal response for nicotine and the deuterated internal standard, precision was determined to be ±1.9%. Nicotine was present in widely varying amounts in the hair of dogs belonging to smokers and was found to be absent from the hair of dogs belonging to non-smokers. In addition to nicotine, nicotine N-oxides, cotinine, nornicotine and nornicotine N-oxide could be detected in the hair of dogs belonging to smokers. The nicotine N-oxides were only observed in methanol extracts suggesting these compounds are not stable to the NaOH digestion process.