| Smoking and health are issues that merit attention. The smoke of cigarette contains more than 5,000 kinds of chemical substances, including a large amount of substances that are hazardous to human health, such as tobacco-specific nitrosamines, particulate- and gas-phase free radicals, polynuclear aromatic hydrocarbons, and other well-known carcinogens. To monitor and reduce these harmful components, it is very necessary to develop methods for the determination of these substances.In our studies, a method has been developed to accurately determine total particulate-phase free radicals in mainstream cigarette smoke. Cambridge filter pad (CFP) was usually employed to collect particulate-phase free radicals in mainstream cigarette smoke, but we found that it was not suitable for the accurate determination of total particulate-phase free radicals in mainstream cigarette smoke. More than 20% of particulate-phase free radicals in mainstream cigarette smoke were observed breaking through Cambridge filter pads in our experiments. A special filter pad with tiny Electron Paramagnetic Resonance (EPR) signal was found out that was suitable for the quantitative analysis of total particulate-phase free radicals in mainstream cigarette smoke. To reduce the difference of test results brought about by using different EPR spectrometer, a new calibration method was developed by using the ratio of EPR signal intensities of 1.1-diphenyl-2-picryl-hydrazyl (DPPH) standard samples to those of high pitch and the spin number of DPPH standard samples in making calibration curve. This method has been validated through intra- and inter-laboratory studies and has shown excellent repeatability and reproducibility.A method has been developed for the determination of the gas-phase free radicals in mainstream cigarette smoke. A trap device for the gas-phase free radicals was developed, which could be easily attached to and removed from commercial linear smoking machine. N-t-butyl-α-phenyl nitrone (PBN) was used to trap the gas-phase free radicals and the formed adduct was detected with EPR technique. To reduce the difference of test results brought about by using different EPR spectrometer, a new calibration method was developed by using the ratio of EPR signal intensities of 2.2.6.6-tetramethyl-piperidinyl-l-oxy (TEMPO) standard samples to those of high pitch and the spin number of DPPH standard samples in making calibration curve. The sampling, condition and selection of cigarettes in the test were accordant with ISO 4387. It was proved that the established methods were sensitive, repeatable, and practicable for the determination of gas-phase free radicals in routine tests.A specific and sensitive method was developed and validated to quantitatively analyze four tobacco-specific nitrosamines (TSNA) in the particulate phase of mainstream cigarette smoke. Cigarette smoke particulate matter was collected according to ISO 4387. The particulate matter was extracted with acetic ether, cleaned up with a Supelclean ENVI-Carb SPE cartridge, concentrated with the protection of nitrogen and analyzed by GC/ ion trap MS with a very-low-flow-loss column (VF-17 MS) and MSn mode using N-nitrosopentyl-3-picolylamine (NNPA) as an internal standard. TSNAs were identified by chromatographic retention time, the match of the spectrum of the standards and the samples, and the consistency of the ratio of the abundance of the ions detected in the standards and the samples. Limits of detection of each TSNA varied from 0.01 ng/cig to 0.06 ng/cig. A linear calibration range for this method is large enough to measure TSNA levels in cigarette smoke. The recovery of each TSNA was from 91.5% to 102.7%. The method achieved excellent reproducibility (RSD: 1.8%-4.8% for intra-assay, 3.4%-7.1% for inter-assay). It also shows no evidence of artifact formation. This method is very suitable for the routine detection of TSNAs in the mainstream cigarette smoke.
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