Secondary Electrospray Ionization Mass Spectrometry for Volatile Analysis: Current Challenges and Emerging Solutions

Secondary electrospray ionization mass spectrometry (SESI-MS) has emerged as a powerful technique for the real-time, non-invasive analysis of volatile organic compounds (VOCs) in complex matrices, such as exhaled breath and microbial volatilomes. However, its transition to routine application is hindered by significant challenges in absolute quantification, unambiguous identification, and standardization. This review provides a comprehensive overview of these limitations and the emerging solutions proposed to overcome them. Matrix effects, including gas-phase ion suppression and C-trap competition, are examined alongside mitigation strategies such as spectral stitching and standard addition. To enhance quantification stability, advanced standard delivery systems and dynamic quality control protocols are evaluated. The identification bottleneck—stemming from the absence of chromatographic separation—is addressed through the use of curated databases and advanced fragmentation techniques, such as incremental quadrupole acquisition to resolve overlapping spectra (IQAROS), to resolve isobaric interferences. Furthermore, the role of chemometrics in extracting biological fingerprints is discussed. Finally, the need for harmonized reporting standards and multicenter validation is emphasized to ensure cross-study reproducibility. Resolving these methodological gaps is essential for the clinical and industrial translation of SESI-MS.