Determination of serum cotinine by isotope dilution liquid chromatography tandem mass spectrometry

Objective To establish a method for measuring serum cotinine by isotope dilution liquid chromatography tandem mass spectrometry （ID-LC/MS/MS）and provide an assay that can be applied to the evaluation of the level of smoke exposure and to the risk analysis of smoking related diseases. Methods Blood samples were collected from 94 apparently healthy subjects from October to December in 2010 and centrifuged, and the sera were separated.Serum samples were mixed with ［D3］-cotinine (as the internal standard) and treated with acetonitriles to precipitate protein.After centrifugation, the supernatants were transferred and evaporated under a stream of nitrogen until dryness and reconstituted with mobile phase.Then the residuals were analyzed by LC/MS/MS system with multiple reaction monitor model; the concentration of cotinine were quantified by the isotope internal standard method and the stand curve was employed with a series of calibration.To estimate the precision of the method, five frozen serum pools were repeatedly analyzed in five runs, and every pool was analyzed in triplicate.In addition, the recovery rates were analyzed with the serum sample added with different levels of standard.The stability of cotinine in serum preserved at room temperature, 4 ℃ and -80 ℃, respectively.Finally, the levels of cotinine of94 healthy subjects were measured to evaluate the distribution of cotinine with different smoke statuses. Results Serum cotinine measured by ID-LC/MS/MS was separated well with few interferences.The correlation coefficients between the peak area ratios and cotinine concentrations were higher than 0.9993.The values of within-run coefficients of variation （CV） of five frozen serum pools (0.68, 48.42, 94.34, 250.95 and 287.04 μg/L) were 2.19%，0.78%，0.75%，0.65% and 0.67％, respectively.The values of total CV were 4.71%，1.40%，1.98%，1.10% and 1.03％, respectively.The limit of detection （LOD） and limit of quantitation （LOQ） were 0.013 and 0.050 μg/L, respectively.The analytical recoveries ranged from 99.22% to 102.67%.The samples could maintain stability within 2 d at room temperature, 7 d at 4 ℃ and 3 months at -80 ℃ resulting the accuracy of measurements from 99.28% to 100.87% and the CV<5%.The levels of cotinine of 94 healthy subjects were measured and shown skewed and leptokurtic distribution.The concentrations of twenty smokers, fourteen former smokers and sixty non-smokers were 116.40 (63.17-241.12), 0.67 (0.15-0.95) and 0.22 (0.15-0.42) μg/L, respectively. Furthermore, the level of cotinine of former smokers (Z=-2.12, P＜0.05) and smokers (Z=-6.67, P<0.001) were statistically higher than non-smokers. Conclusions An ID-LC/MS/MS method for serum cotinine detection has been established.It is hoped that the method will be applied to the assessment of smoke exposure and its association with the risks of smoking related diseases since it is simple, specific, precise, sensitive and accurate.（Chin J Lab Med, 2012, 35:333-338） Key words: Smoke; Cotinine; Isotope dilution mass spectrometry; Liquid chromatography tandem mass spectrometry