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The detection of volatile organic compounds (VOCs) in human breath may be useful for routine clinical diagnosis of several diseases in a non-invasive manner. Traditional methods of breath analysis have some technical limitations. Membrane extraction with a sorbent interface (MESI), however, has many advantages over current methods, including good selectivity and sensitivity, and is well suited for breath analysis.The aim of this project was to develop a simple and reproducible sampling device and method based on the MESI system for breath analysis. The feasibility and validity of the MESI system was tested with real human breath samples. The use of breath CO
2
as an internal standard for the analysis of breath VOCs is an effective method to solve the difficulties associated with variations in the target analyte concentrations in a sample, which are attributed to mass losses and different breathing patterns of different subjects. In this study, the concentration of breath acetone was successfully expressed normalized to CO
2
as in the alveolar air. Breath acetone of healthy males and females profiled at different times of the day was plotted using the MESI system, and results are consistent with literature. This technique can be used for monitoring breath acetone concentrations of diabetic patients and for applications with other biomarker monitoring.