Secondary electrospray ionisation high-resolution mass spectrometry (SESI-HRMS) is a powerful, non
invasive tool for real-time breath analysis. However, instrumental drift, capillary degradation and other
technical variations compromise clinical applications, especially for long-term studies, which require
robust quality control (QC) methodologies. In this work, we present the use of an evaporation chamber
based reference gas system to monitor SESI-MS system performance over almost two years. Targeted
QC measurements with deuterated butyric acid were recorded three times on each measurement day.
Analytical stability was systematically quantified across both positive and negative ion modes. In
a standalone continuous characterization, the reference gas generator delivered a stable signal over
several hours. A Linear Mixed-Effects Model (LMM) was constructed to evaluate the influence of time-of
day on instrument performance. This revealed significant effects: positive mode signal intensity
progressively increased throughout the day, whereas for negative mode, an initial drop in signal intensity
and subsequent stabilisation was observed. Furthermore, the longitudinal QC framework successfully
tracked capillary performance over time, providing objective performance metrics to retrospectively
evaluate the impact of targeted hardware maintenance on system performance. The implementation of
a structured QC protocol provides a valid methodology for long-term SESI-HRMS performance
monitoring, ensuring data integrity and supporting the translation of SESI-HRMS into large-scale clinical
applications.