Abstract:

The analytical method of nicotine and cotinine in human urine with hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) was established. After the urine sample containing nicotine-d₄ and cotinine-d₃ isotope internal standards being diluted with water, the filtrate was introduced into ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Separation was performed on an ACQUITY UPLC^® BEH HILIC column (50 mm×3.0 mm, 1.7 μ m), in which methanol and 0.1% (v/v) ammonia were used as the mobile phases with isocratic elution at 0.2 mL/min of flow rate. Positive ion scan mode was used for mass spectrometry measurement and calibration curves were plotted for quantification determination. A good linearity could be obtained in the range of 1.0-1000 μ g/L for nicotine and cotinine with the linear coefficients of 0.9949 and 0.9958, respectively. The limits of detection of nicotine and cotinine were 0.082 μ g/L and 0.077 μ g/L, and the limits of quantification were 0.27 μ g/L and 0.26 μ g/L, respectively. The recoveries of the spiked urine samples were 90.4%-103.5% and 93.0%-104.6%, and the relative standard deviations (RSDs) were 4.80%-6.21% and 4.22%-7.15% for nicotine and cotinine respectively. The established method was applied to the analyis of 200 urine samples. Based on the investigation information of the urine of the smoking people, the nicotine contents were 26.68-854.30 μ g/L, and the cotinine contents were 36.66-1191.18 μ g/L (n=86, M_nicotine=76.00 μ g/L, M_cotinine=83.52 μ g/L, M:median); of the nonsmoking people, the nicotine contents were 5.08-69.66 μ g/L, and the cotinine contents were 3.16-28.21 μ g/L (n=114, M_nicotine=7.53 μ g/L, M_cotinine=3.79 μ g/L). The method is simple, sensitive and rapid. It is suitable for batch analysis of nicotine and cotinine in urine, and it can meet the requirement of evaluating the human tobacco exposure.

Key words: cotinine, hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS), isotope dilution, nicotine, urine