An effective protocol for quenching should take certain factors into consideration to achieve rapid and thorough inhibition of intracellular metabolic reactions . Studies have been performed to evaluate the performance of different quenching protocols, including cold isotonic saline (0.9% NaCl) , chilled acetonitrile (at -40°C) , cold methanol (60%, at -40°C) containing buffer salts (e.g., ammonium bicarbonate , NaCl , HEPES , ammonium carbonate ), ice-cold phosphate-buffered saline (PBS) , LN2 , and hot air treatments . In fact, some of these above quenching methods were designed for MS metabolomics studies of bulk cell samples, and cold methanol and acetonitrile have been utilized with Pico-ESI-MS and MALDI-MS techniques, respectively, for in single cell studies. Although these quenching methods have demonstrated their efficacy in halting enzymatic activity and preserving cellular metabolites, each approach has its own limitations: organic solvents could lead to metabolite leakage and cell membrane damage ; using solutions containing nonvolatile salts can severely impact MS analysis due to matrix effect, which leads to ion suppression , reduced sensitivity, inaccurate quantification of analytes, and ion signal interference.