D2-nicotine, D2-cotinine, D2-trans-3′-hydroxycotinine, non-deuterated (D0)-cotinine and D0-trans-3′-hydroxycotinine were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) by modification of a previously described method[21]. Changes to the method included: the addition of D9-trans-3′-hydroxycotinine (a gift from Dr, Peyton Jacob, University of California, San Francisco) to the plasma samples as an internal standard and elution of the solid phase extraction column with methanol containing 2% ammonium hydroxide (MeOH/base). An aliquot was removed for nicotine analysis and the remainder was evaporated to dryness, and resuspended in 1/10th the volume of MeOH/base for cotinine and trans 3′-hydroxycotinine analysis. LC/MS/MS for nicotine was as previously described [21], whereas the simultaneous analysis of cotinine and trans 3′-hydroxycotinine required a mobile phase of 95% acetonitrile, 4% water, 1% formic acid, flow rate, 20ul/min. Cotinine eluted at 2.53 min and trans 3′-hydroxycotinine eluted at 2.65min. The mass transitions monitored for D-and D9-trans-3′-hydroxycotinine were m/z 193 → 80 and m/z 202 → 84, respectively. The limits of quantitation for D2-nicotine, D2-cotinine, D2 trans 3′-hydroxycotinine were 0.25, 0.25 and 0.6 ng/ml plasma. D2-nicotine, D2-cotinine, and D0-cotinine plasma concentrations were determined
