Abstract: The nicotine,cotinine,trans-3'-hydroxycotinine,and their corresponding glucosides,as well as nornicotine,norcotinine,cotinine-N-oxide and nicotine-N'-oxide in the urine samples from ten smokers and five passive smokers were determined by liquid chromatography with tandem mass spectrometry(LC-MS/MS),using deuterium-labeled nicotine,cotinine,and trans-3'-hydroxycotinine as internal standards.The compounds were separated on an XDBC₁₈ column by gradient elution of the mixture of 10 mmol/L ammonium acetate water solution(pH 6.8) and methanol,a mobile phase,at a flow rate of 1mL/min.The results showed that:1) the recoveries of the analytes ranged from 78% to 116% with the relative standard deviation(RSD) of 2.1% to 17.0%;2) the contents of nicotine and its metabolites in the urine samples from smokers who smoked 20 cigarettes of the same brand everyday differed obviously;3) nicotine,cotinine and trans-3'-hydroxycotinine were detectable in all urine samples from passive smokers,cotinine-N-glucoside,nornicotine and nicotine-N'-oxide were detectable in some of the samples from passive smokers at slightly lower levels;while all of them were far lower than these in smoker's urine samples.Nicotine,cotinine and trans-3'-hydroxycotinine in human urine might be used as the biomarkers of tobacco smoke exposure.