BACKGROUND-AIM:
Heated tobacco products (HTPs) are an emerging tobacco product that generate nicotine-containing aerosol for consumer inhalation by heating rather than burning tobacco. Given that certain HTPs have been legally introduced to the market in Taiwan as of October 2025, it is need to analyze their aerosols for components that may pose risks to human health. Nicotine and tobacco-specific nitrosamines (TSNAs) are well known the harmful and potentially harmful constituents in tobacco products and their mainstream and are also regulated components in Taiwan. The aim of this study is to establish an analytical method for determining the levels of nicotine and TSNAs in the mainstream aerosol of HTPs.

METHODS:
The pre-conditioning, environmental control, puffing parameters, and aerosol collection procedures for HTPs were conducted in accordance with ISO 5501. Mainstream aerosols were collected using a linear smoking machine onto 44 mm glass fiber filter pads. For nicotine analysis, the filter pads were extracted by shaking with 20 mL of isopropanol containing an internal standard, followed by analysis using gas chromatography with flame ionization detection. For TSNAs analysis, the pads were extracted with 20 mL of 100 mM ammonium acetate solution containing isotopically labeled internal standards, filtered and analyzed using liquid chromatography-tandem mass spectrometry. Both methods were validated using the commercially available product. Two concentration levels of reference standards were spiked to the filter pads after completion of the smoking process. The subsequent extraction and analysis followed the established procedures.

RESULTS:
The results showed that the calibration curve for nicotine ranged from 0.02 to 0.5 mg/mL, and the calibration curve for TSNAs ranged from 0.3 to 50 ng/mL, with correlation coefficients (r) greater than 0.99, indicating good linearity. The intra-day and inter-day average recoveries for nicotine ranged from 88.3% to 97.9%, and the coefficients of variation (CV) between 0.8% and 2.2%. The intra-day and inter-day average recoveries for TSNAs ranged from 89.8% to 116.1%, with CV values between 0.5% and 8.2%, demonstrating good accuracy and precision of the analytical method.

CONCLUSIONS:
The analytical method for nicotine and TSNAs in the mainstream aerosol of HTPs established in this study is appropriate and stable. Due to the current lack of certified reference materials for HTPs internationally, method validation and quality assurance remain challenging. Therefore, continued efforts are needed to seek certified reference material to ensure the accuracy of results. Additionally, the development of analytical methods for other harmful constituents in the mainstream aerosol of HTPs will also be ongoing. ACKNOWLEDGEMENTS This work was funded by the Health Promotion Administration, Ministry of Health and Welfare. The content of this research may not represent the opinion of Ministry of Health and Welfare.