Simultaneous determination of nicotine and its nine metabolites in human urine by LC-MS-MS

A method based on liquid chromatography tandem mass spectrometry was developed for the direct determination of nicotine, cotinine, trans-3'-hydroxycotinine, their corresponding glucuronide conjugates as well as nornicotine, norcotinine, cotinine-N-oxide and nicotine-N'-oxide in the urine of smokers. The assay only involves centrifugation and filtration of diluted urine. The analysis was performed on a C18 reversed-phase column using a gradient of 10 mM ammonium acetate, pH 6.8, and methanol as mobile phase at a flow rate of 1 mL min(-1). Nicotine-methyl-d(3), Cotinine-methyl-d(3) and trans-3'-hydroxycotinine-methyl-d(3) were used as internal standards. Precisions (RSD) for all the analytes at three levels were between 2.1 and 17.0%. Recoveries for nicotine and nine nicotine metabolites ranged from 78.4 to 115.6%. The described method was suitable for determining the nicotine dose in large-scale human biomonitoring studies.