In order to accurately determine pyrazines and pyridines in saliva of tobacco smokers, a method of gas chromatography-positive chemical ionization-time of flight mass spectrometry (GC-PCI-TOF MS) was developed. The pre-treatment method of sample, chromatogphic condition and MS condition were optimized. Saliva samples were extracted with dichloromethane by vortex mixing and cleaned up by primary secondary amine (PSA), then detected by GC-PCI-TOF MS. Retention time, precise mass data of characteristic ions and fragmentation patterns of characteristic fragment ions were employed to qualitative identification, external standard method was used for quantitative determination. The results showed that: 1) The average of extraction yield of 11 pyrazines and pyridines was 97.7% when 5 mL saliva sample was extracted by dichloromethane two times. 2) The matrix effects were studied by comparing the pyrazines and pyridines peak response in matrix standard solution and matrix-free standard solution, which performed with no significance. 3) Mass number of characteristic ions measured was in good agreement with predicted value, and the error was less than 3.12×10-6. Besides, predominant molecular ion and adduct molecular ion peak were obtained in PCI mode with less fragment ions formation. The mass spectrum was simple and clear, which was easy to identify. Given the above, the accuracy of qualitative analysis for low concentrations of pyrazines and pyridines in saliva could be significantly improved. 4) The linear correlation coefficients were above 0.999 1, the limits of detection (LODs) ranged from 0.9 μg/L to 2.4 μg/L, the limits of quantitation (LOQs) ranged from 3.0 μg/L to 7.9 μg/L, and the recoveries ranged from 85% to 104% at three spiked levels. The quantitative method was proved to be high accuracy and high sensitivity. 5) The detection results of saliva of tobacco smokers showed that the main pyrazines and pyridines in smokers’ saliva were pyridine, 2-methylpyrazine and dimethylpyrazines, and the contents of pyridine and 2-methylpyrazine in smokers’ saliva was differed by cigarette type. Low concentration of trimethylpyrazine and tetramethylpyrazine were detected in specific saliva samples of tobacco smokers. 3-Ethylpyridine and 5-ethyl-2-methylpyridine were not found in experimental tobacco smokers’ saliva sample. 6) Pyrazines and pyridines’ contents in different smokers’ saliva of 6 kind cigarettes were analyzed using principal component analysis which can distinguish different types of cigarette. Further analysis indicated that there was a significant variance-(P<0.05) between the pyrazines and pyridines in saliva among the 6 different kinds of cigarette, which might be a significantly characteristic of cigarette type. The method is simple, rapid and accuracy, which is superior in analyzing low level of pyrazines and pyridines in saliva of tobacco smokers and similar complex matrix. The study result will also offer accurate and scientific reference on cigarette product development. [ABSTRACT FROM AUTHOR]