Analysis of [3′,3′-d₂]-nicotine and [3′,3′-d₂]-cotinine by capillary liquid chromatography-electrospray tandem mass spectrometry

A selective and sensitive LC/MS/MS assay was developed for the quantification of d₂-nicotine and d₂-cotinine in plasma of current and past smokers administered d₂-nicotine. After solid phase extraction and liquid-liquid extraction, HPLC separation was achieved on a capillary hydrophilic interaction chromatography phase column. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated for d₂-nicotine (0.03-6.0 ng/ml plasma) and d₂-cotinine (0.15-25 ng/ml plasma). The lower limits of quantitation were 0.15 ng/ml and 0.25 ng/ml for d₂-nicotine and d₂-cotinine, respectively. The coefficient of variation was 3.7% for d₂-nicotine and 2.5% for d₂-cotinine. The method was applied to two ongoing studies of d₂-nicotine metabolism in prior and current smokers. Preliminary analysis of a subset of subjects from these studies detected a significantly lower rate of nicotine conversion to cotinine by past smokers compared to current smokers.