Protocol for the untargeted analysis of volatile metabolites in the headspace of biological samples using Thin-Film Microextraction coupled to GC–MS

• TFME meshes are green and reusable devices for extracting a wide range of analytes. • TFME can be used to analyze complex biological matrices. • The protocol provides step-by-step guidance on the TFME application. Monitoring volatile organic compounds released by pathogenic microorganisms or cancer cells in biological samples, such as blood, urine, saliva, bronchoalveolar lavage (BAL), or breath, can help detect their presence, enabling rapid diagnosis and targeted therapy. However, the complexity of biological matrices and the trace concentrations of target analytes necessitate the use of effective preconcentration techniques for accurate analysis. One of these techniques is thin-film microextraction (TFME), which improves extraction efficiency compared to widely used SPME. TFME offers a cost-effective and green extraction of complex biological samples due to reusable materials, solvent-free extraction, and thermal desorption. The first part of this work provides an overview of the parameters considered for the optimization and their impact on extraction efficiency, including the type of sorbent, extraction mode, temperature and time of extraction, sample volume, and desorption conditions. The applications of TFME in the targeted and untargeted analysis of biological samples are also discussed and exemplified in the pioneering and exceptional studies demonstrating the capabilities of TFME. The protocol described here introduces the TFME-GC–MS method for untargeted analysis of VOCs in the headspace of biological samples, providing a step-by-step guide with the subsequent troubleshooting section. The procedure underwent a comprehensive evaluation, considering time, estimated cost, safety, and, most importantly, the method's greenness, resulting in a score of 0.72 on the AGREE scale and 0.60 on the AGREEprep scale.