Quantifying Nicotine and Cotinine in Mice

The plasma concentration of nicotine and its principal metabolite cotinine were measured from the 14 days JUUL/TS-exposed mice by LCMS/MS analysis using cotinine-d3 (MilliporeSigma, St. Louis, MO, USA) as internal standard (IS) following a previously published method [23 (link)]. In brief, samples were prepared by protein precipitation of 25 µl mouse plasma using acetonitrile at a 1:8 ratio. The mass spectrometer was operated in positive polarity under the multiple reaction monitoring mode using the electrospray ionization technique. The transitions of m/z 163.2 → 132.1, 177.2 → 98.0, and 180.2 → 101.2 were used to measure the nicotine, cotinine, and IS, respectively. The elution of nicotine (MilliporeSigma), cotinine (MilliporeSigma), and IS were at 1.89, 1.77, and 1.76 min, respectively. This was achieved with a mobile gradient phase consisting of 5 mM ammonium bicarbonate, acetonitrile, and methanol (3:1, v/v) at a 0.3 ml/min flow rate on a Kinetex EVO C18 column (Phenomenex, Torrance, CA, USA).